Publications by authors named "Michael Freissmuth"

158 Publications

Assessing the Accuracy of Sales Forecasts Submitted by Pharmaceutical Companies Applying for Reimbursement in Austria.

Front Pharmacol 2021 13;12:726758. Epub 2021 Aug 13.

Institute of Pharmacology, Centre of Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria.

Reimbursement decisions on new medicines require an assessment of their value. In Austria, when applying for reimbursement of new medicines, pharmaceutical companies are also obliged to submit forecasts of future sales. We systematically examined the accuracy of these pharmaceutical sales forecasts and hence the usefulness of these forecasts for reimbursement evaluations. We retrospectively analyzed reimbursement applications of 102 new drugs submitted between 2005 and 2014, which were accepted for reimbursement outside of hospitals, and for which actual reimbursed sales were available for at least 3 years. The main outcome variable was the accuracy ratio, defined as the ratio of forecasted sales submitted by pharmaceutical companies when applying for reimbursement to actual sales from reimbursement data. The median accuracy ratio [95% confidence interval] was 1.33 [1.03; 1.74, range 0.15-37.5], corresponding to a median overestimation of actual sales by 33%. Forecasts of actual sales for 55.9% of all examined products either overestimated actual sales by more than 100% or underestimated them by more than 50%. The accuracy of sales forecasts did not show systematic change over the analyzed decade nor was it discernibly influenced by reimbursement status (restricted or unrestricted), the degree of therapeutic benefit, or the therapeutic area of the pharmaceutical product. Sales forecasts of drugs with a higher degree of innovation and those within a dynamic market tended to be slightly more accurate. The majority of sales forecasts provided by applicants for reimbursement evaluations in Austria were highly inaccurate and were on average too optimistic. This is in line with published results for other jurisdictions and highlights the need for caution when using such forecasts for reimbursement procedures.
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http://dx.doi.org/10.3389/fphar.2021.726758DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8414520PMC
August 2021

Plant-Derived Cyclotides Modulate κ-Opioid Receptor Signaling.

J Nat Prod 2021 08 26;84(8):2238-2248. Epub 2021 Jul 26.

Center for Physiology and Pharmacology, Institute of Pharmacology, Medical University of Vienna, 1090 Vienna, Austria.

Cyclotides are plant-derived disulfide-rich peptides comprising a cyclic cystine knot, which confers remarkable stability against thermal, proteolytic, and chemical degradation. They represent an emerging class of G protein-coupled receptor (GPCR) ligands. In this study, utilizing a screening approach of plant extracts and pharmacological analysis we identified cyclotides from to be ligands of the κ-opioid receptor (KOR), an attractive target for developing analgesics with reduced side effects and therapeutics for multiple sclerosis (MS). This prompted us to verify whether [T20K]kalata B1, a cyclotide in clinical development for the treatment of MS, is able to modulate KOR signaling. T20K bound to and fully activated KOR in the low μM range. We then explored the ability of T20K to allosterically modulate KOR. Co-incubation of T20K with KOR ligands resulted in positive allosteric modulation in functional cAMP assays by altering either the efficacy of dynorphin A or the potency and efficacy of U50,488 (a selective KOR agonist), respectively. In addition, T20K increased the basal response upon cotreatment with U50,488. In the bioluminescence resonance energy transfer assay T20K negatively modulated the efficacy of U50,488. This study identifies cyclotides capable of modulating KOR and highlights the potential of plant-derived peptides as an opportunity to develop cyclotide-based KOR modulators.
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http://dx.doi.org/10.1021/acs.jnatprod.1c00301DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8406418PMC
August 2021

Concomitant oral intake of purified clinoptilolite tuff (G-PUR) reduces enteral lead uptake in healthy humans.

Sci Rep 2021 07 20;11(1):14796. Epub 2021 Jul 20.

Department of Clinical Pharmacology, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria.

Lead exposure can cause substantial organ damage. Enteral lead absorption may be reduced by concomitant intake of clinoptilolite tuff, a zeolite from natural sources. This study aimed to assess the effect of purified clinoptilolite tuff (G-PUR) on enteral lead uptake in adults using stable lead isotope Pb as a tracer. In this randomized, placebo-controlled, double-blind, parallel-group study, 42 healthy participants were randomized to receive oral G-PUR 2.0 g, 2 * 2.0 g, or placebo, together with 2.5 µg of Pb in water. The enrichment of Pb caused by the tracer in blood and urine was measured by mass spectrometry. G-PUR was well tolerated. The mean maximum Pb enrichment of 0.505% of total blood lead was significantly higher (p < 0.0001) in the placebo group compared to G-PUR 2.0 g (0.073%) or G-PUR 2 * 2.0 g (0.057%) group. Normalized Pb AUC was 86.5, 11.9, and 8.5% * h without and with G-PUR 2.0 g, and G-PUR 2 * 2.0 g, respectively (p < 0.0001 vs. placebo). This smaller Pb exposure was paralleled by a reduced urinary excretion in subjects receiving G-PUR. Concomitant oral intake of purified clinoptilolite tuff reduced enteral uptake of Pb in healthy humans by approximately 90%. The reduced bioavailability is demonstrable by a decrease of Pb tracer enrichment in blood and urine.Trial registration: clinicaltrials.gov identifier: NCT04138693, registered 24/10/2019.
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http://dx.doi.org/10.1038/s41598-021-94245-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8292361PMC
July 2021

Extracellular loops of the serotonin transporter act as a selectivity filter for drug binding.

J Biol Chem 2021 07 9;297(1):100863. Epub 2021 Jun 9.

Institute of Pharmacology and the Gaston H. Glock Research Laboratories for Exploratory Drug Development, Center of Physiology and Pharmacology, Medical University of Vienna, Austria.

The serotonin transporter (SERT) shapes serotonergic neurotransmission by retrieving its eponymous substrate from the synaptic cleft. Ligands that discriminate between SERT and its close relative, the dopamine transporter DAT, differ in their association rate constant rather than their dissociation rate. The structural basis for this phenomenon is not known. Here we examined the hypothesis that the extracellular loops 2 (EL2) and 4 (EL4) limit access to the ligand-binding site of SERT. We employed an antibody directed against EL4 (residues 388-400) and the antibody fragments 8B6 scFv (directed against EL2 and EL4) and 15B8 Fab (directed against EL2) and analyzed their effects on the transport cycle of and inhibitor binding to SERT. Electrophysiological recordings showed that the EL4 antibody and 8B6 scFv impeded the initial substrate-induced transition from the outward to the inward-facing conformation but not the forward cycling mode of SERT. In contrast, binding of radiolabeled inhibitors to SERT was enhanced by either EL4- or EL2-directed antibodies. We confirmed this observation by determining the association and dissociation rate of the DAT-selective inhibitor methylphenidate via electrophysiological recordings; occupancy of EL2 with 15B8 Fab enhanced the affinity of SERT for methylphenidate by accelerating its binding. Based on these observations, we conclude that (i) EL4 undergoes a major movement during the transition from the outward to the inward-facing state, and (ii) EL2 and EL4 limit access of inhibitors to the binding of SERT, thus acting as a selectivity filter. This insight has repercussions for drug development.
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http://dx.doi.org/10.1016/j.jbc.2021.100863DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8253976PMC
July 2021

Handling of intracellular K determines voltage dependence of plasmalemmal monoamine transporter function.

Elife 2021 06 1;10. Epub 2021 Jun 1.

Institute of Pharmacology and the Gaston H. Glock Research Laboratories for Exploratory Drug Development, Center of Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria.

The concentrative power of the transporters for dopamine (DAT), norepinephrine (NET), and serotonin (SERT) is thought to be fueled by the transmembrane Na gradient, but it is conceivable that they can also tap other energy sources, for example, membrane voltage and/or the transmembrane K gradient. We have addressed this by recording uptake of endogenous substrates or the fluorescent substrate APP(4-(4-dimethylamino)phenyl-1-methylpyridinium) under voltage control in cells expressing DAT, NET, or SERT. We have shown that DAT and NET differ from SERT in intracellular handling of K. In DAT and NET, substrate uptake was voltage-dependent due to the transient nature of intracellular K binding, which precluded K antiport. SERT, however, antiports K and achieves voltage-independent transport. Thus, there is a trade-off between maintaining constant uptake and harvesting membrane potential for concentrative power, which we conclude to occur due to subtle differences in the kinetics of co-substrate ion binding in closely related transporters.
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http://dx.doi.org/10.7554/eLife.67996DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8192120PMC
June 2021

Binding and neutralization of C. difficile toxins A and B by purified clinoptilolite-tuff.

PLoS One 2021 27;16(5):e0252211. Epub 2021 May 27.

GLOCK Health, Science and Research G.m.b.H., Deutsch-Wagram, Austria.

Clostridioides difficile (C. difficile) infection is a major public health problem worldwide. The current treatment of C. difficile-associated diarrhea relies on the use of antibacterial agents. However, recurrences are frequent. The main virulence factors of C. difficile are two secreted cytotoxic proteins toxin A and toxin B. Alternative research exploring toxin binding by resins found a reduced rate of recurrence by administration of tolevamer. Hence, binding of exotoxins may be useful in preventing a relapse provided that the adsorbent is innocuous. Here, we examined the toxin binding capacity of G-PUR®, a purified version of natural clinoptilolite-tuff. Our observations showed that the purified clinoptilolite-tuff adsorbed clinically relevant amounts of C. difficile toxins A and B in vitro and neutralized their action in a Caco-2 intestinal model. This conclusion is based on four independent sets of findings: G-PUR® abrogated toxin-induced (i) RAC1 glucosylation, (ii) redistribution of occludin, (iii) rarefaction of the brush border as visualized by scanning electron microscopy and (iv) breakdown of the epithelial barrier recorded by transepithelial electrical resistance monitoring. Finally, we confirmed that the epithelial monolayer tolerated G-PUR® over a wide range of particle densities. Our findings justify the further exploration of purified clinoptilolite-tuff as a safe agent in the treatment and/or prevention of C. difficile-associated diarrhea.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0252211PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8158989PMC
May 2021

Constitutive Endocytosis of the Neuronal Glutamate Transporter Excitatory Amino Acid Transporter-3 Requires ARFGAP1.

Front Physiol 2021 10;12:671034. Epub 2021 May 10.

Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria.

The eukaryotic endocytic pathway regulates protein levels available at the plasma membrane by recycling them into specific endosomal compartments. ARFGAP1 is a component of the coat protein I (COPI) complex but it also plays a role in promoting adapter protein-2 (AP-2) mediated endocytosis. The excitatory amino acid transporter-3 (EAAT3) mediates the reuptake of glutamate from the synaptic cleft to achieve rapid termination of synaptic transmission at glutamatergic synapses. In this study, we identified two interacting proteins of EAAT3 by mass spectrometry (MS) ARFGAP1 and ARF6. We explored the role of ARFGAP1 and ARF6 in the endocytosis of EAAT3. Our data revealed that ARFGAP1 plays a role in the recycling of EAAT3, by utilizing its GTPase activating protein (GAP) activity and ARF6 acting as the substrate. ARFGAP1 promotes cargo sorting of EAAT3 a single phenylalanine residue (F508) located at the C-terminus of the transporter. ARFGAP1-promoted AP-2 dependent endocytosis is abolished upon neutralizing F508. We utilized a heterologous expression system to identify an additional motif in the C-terminus of EAAT3 that regulates its endocytosis. Impairment in endocytosis did not affect somatodendritic targeting in cultured hippocampal neurons. Our findings support a model where endocytosis of EAAT3 is a multifactorial event regulated by ARFGAP1, occurring the C-terminus of the transporter, and is the first study to examine the role of ARFGAP1 in the endocytosis of a transport protein.
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http://dx.doi.org/10.3389/fphys.2021.671034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8141794PMC
May 2021

Gene therapy restores dopamine transporter expression and ameliorates pathology in iPSC and mouse models of infantile parkinsonism.

Sci Transl Med 2021 05;13(594)

Gene Transfer Technology Group, EGA-Institute for Women's Health, University College London, London, WC1E 6HX, UK.

Most inherited neurodegenerative disorders are incurable, and often only palliative treatment is available. Precision medicine has great potential to address this unmet clinical need. We explored this paradigm in dopamine transporter deficiency syndrome (DTDS), caused by biallelic loss-of-function mutations in , encoding the dopamine transporter (DAT). Patients present with early infantile hyperkinesia, severe progressive childhood parkinsonism, and raised cerebrospinal fluid dopamine metabolites. The absence of effective treatments and relentless disease course frequently leads to death in childhood. Using patient-derived induced pluripotent stem cells (iPSCs), we generated a midbrain dopaminergic (mDA) neuron model of DTDS that exhibited marked impairment of DAT activity, apoptotic neurodegeneration associated with TNFα-mediated inflammation, and dopamine toxicity. Partial restoration of DAT activity by the pharmacochaperone pifithrin-μ was mutation-specific. In contrast, lentiviral gene transfer of wild-type human complementary DNA restored DAT activity and prevented neurodegeneration in all patient-derived mDA lines. To progress toward clinical translation, we used the knockout mouse model of DTDS that recapitulates human disease, exhibiting parkinsonism features, including tremor, bradykinesia, and premature death. Neonatal intracerebroventricular injection of human using an adeno-associated virus (AAV) vector provided neuronal expression of human DAT, which ameliorated motor phenotype, life span, and neuronal survival in the substantia nigra and striatum, although off-target neurotoxic effects were seen at higher dosage. These were avoided with stereotactic delivery of AAV2.SLC6A3 gene therapy targeted to the midbrain of adult knockout mice, which rescued both motor phenotype and neurodegeneration, suggesting that targeted AAV gene therapy might be effective for patients with DTDS.
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http://dx.doi.org/10.1126/scitranslmed.aaw1564DOI Listing
May 2021

Tropane-Based Ibogaine Analog Rescues Folding-Deficient Serotonin and Dopamine Transporters.

ACS Pharmacol Transl Sci 2021 Apr 28;4(2):503-516. Epub 2020 Aug 28.

Molecular Targets and Medications Discovery Branch, National Institute on Drug Abuse, Intramural Research Program, Baltimore, Maryland 21224, United States.

Missense mutations that give rise to protein misfolding are rare, but collectively, defective protein folding diseases are consequential. Folding deficiencies are amenable to pharmacological correction (pharmacochaperoning), but the underlying mechanisms remain enigmatic. Ibogaine and its active metabolite noribogaine correct folding defects in the dopamine transporter (DAT), but they rescue only a very limited number of folding-deficient DAT mutant proteins, which give rise to infantile Parkinsonism and dystonia. Herein, a series of analogs was generated by reconfiguring the complex ibogaine ring system and exploring the structural requirements for binding to wild-type transporters, as well as for rescuing two equivalent synthetic folding-deficient mutants, SERT-PGAA and DAT-PGAA. The most active tropane-based analog () was also an effective pharmacochaperone in harboring the DAT-PGAA mutation and rescued 6 out of 13 disease-associated human DAT mutant proteins . Hence, a novel lead pharmacochaperone has been identified that demonstrates medication development potential for patients harboring DAT mutations.
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http://dx.doi.org/10.1021/acsptsci.0c00102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8033614PMC
April 2021

Descriptors of Secondary Active Transporter Function and How They Relate to Partial Reactions in the Transport Cycle.

Membranes (Basel) 2021 Mar 3;11(3). Epub 2021 Mar 3.

Center of Physiology and Pharmacology, Institute of Pharmacology and the Gaston H. Glock Research Laboratories for Exploratory Drug Development, Medical University of Vienna, 1090 Vienna, Austria.

Plasmalemmal solute carriers (SLC) gauge and control solute abundance across cellular membranes. By virtue of this action, they play an important role in numerous physiological processes. Mutations in genes encoding the SLCs alter amino acid sequence that often leads to impaired protein function and onset of monogenic disorders. To understand how these altered proteins cause disease, it is necessary to undertake relevant functional assays. These experiments reveal descriptors of SLC function such as the maximal transport velocity (V), the Michaelis constant for solute uptake (K), potencies for inhibition of transporter function (IC/EC), and many more. In several instances, the mutated versions of different SLC transporters differ from their wild-type counterparts in the value of these descriptors. While determination of these experimental parameters can provide conjecture as to how the mutation gives rise to disease, they seldom provide any definitive insights on how a variant differ from the wild-type transporter in its operation. This is because the experimental determination of association between values of the descriptors and several partial reactions a transporter undergoes is casual, but not causal, at best. In the present study, we employ kinetic models that allow us to derive explicit mathematical terms and provide experimental descriptors as a function of the rate constants used to parameterize the kinetic model of the transport cycle. We show that it is possible to utilize these mathematical expressions to deduce, from experimental outcomes, how the mutation has impinged on partial reactions in the transport cycle.
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http://dx.doi.org/10.3390/membranes11030178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8001282PMC
March 2021

Corrigendum to "Functional and Biochemical Consequences of Disease Variants in Neurotransmitter Transporters: A Special Emphasis on Folding and Trafficking Deficits" [Pharmacology & Therapeutics 222 (2021) 107785].

Pharmacol Ther 2021 Sep 11;225:107816. Epub 2021 Feb 11.

Institute of Pharmacology and the Gaston H. Glock Research Laboratories for Exploratory Drug Development, Center of Physiology and Pharmacology, Medical University of Vienna, Vienna A-1090, Austria. Electronic address:

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http://dx.doi.org/10.1016/j.pharmthera.2021.107816DOI Listing
September 2021

Functional and Biochemical Consequences of Disease Variants in Neurotransmitter Transporters: A Special Emphasis on Folding and Trafficking Deficits.

Pharmacol Ther 2021 06 10;222:107785. Epub 2020 Dec 10.

Institute of Pharmacology and the Gaston H. Glock Research Laboratories for Exploratory Drug Development, Center of Physiology and Pharmacology, Medical University of Vienna, A-1090 Vienna, Austria. Electronic address:

Neurotransmitters, such as γ-aminobutyric acid, glutamate, acetyl choline, glycine and the monoamines, facilitate the crosstalk within the central nervous system. The designated neurotransmitter transporters (NTTs) both release and take up neurotransmitters to and from the synaptic cleft. NTT dysfunction can lead to severe pathophysiological consequences, e.g. epilepsy, intellectual disability, or Parkinson's disease. Genetic point mutations in NTTs have recently been associated with the onset of various neurological disorders. Some of these mutations trigger folding defects in the NTT proteins. Correct folding is a prerequisite for the export of NTTs from the endoplasmic reticulum (ER) and the subsequent trafficking to their pertinent site of action, typically at the plasma membrane. Recent studies have uncovered some of the key features in the molecular machinery responsible for transporter protein folding, e.g., the role of heat shock proteins in fine-tuning the ER quality control mechanisms in cells. The therapeutic significance of understanding these events is apparent from the rising number of reports, which directly link different pathological conditions to NTT misfolding. For instance, folding-deficient variants of the human transporters for dopamine or GABA lead to infantile parkinsonism/dystonia and epilepsy, respectively. From a therapeutic point of view, some folding-deficient NTTs are amenable to functional rescue by small molecules, known as chemical and pharmacological chaperones.
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http://dx.doi.org/10.1016/j.pharmthera.2020.107785DOI Listing
June 2021

The Creatine Transporter Unfolded: A Knotty Premise in the Cerebral Creatine Deficiency Syndrome.

Front Synaptic Neurosci 2020 23;12:588954. Epub 2020 Oct 23.

Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria.

Creatine provides cells with high-energy phosphates for the rapid reconstitution of hydrolyzed adenosine triphosphate. The eponymous creatine transporter (CRT1/SLC6A8) belongs to a family of solute carrier 6 (SLC6) proteins. The key role of CRT1 is to translocate creatine across tissue barriers and into target cells, such as neurons and myocytes. Individuals harboring mutations in the coding sequence of the human CRT1 gene develop creatine transporter deficiency (CTD), one of the pivotal underlying causes of cerebral creatine deficiency syndrome. CTD encompasses an array of clinical manifestations, including severe intellectual disability, epilepsy, autism, development delay, and motor dysfunction. CTD is characterized by the absence of cerebral creatine, which implies an indispensable role for CRT1 in supplying the brain cells with creatine. CTD-associated variants dramatically reduce or abolish creatine transport activity by CRT1. Many of these are point mutations that are known to trigger folding defects, leading to the retention of encoded CRT1 proteins in the endoplasmic reticulum and precluding their delivery to the cell surface. Misfolding of several related SLC6 transporters also gives rise to detrimental pathologic conditions in people; e.g., mutations in the dopamine transporter induce infantile parkinsonism/dystonia, while mutations in the GABA transporter 1 cause treatment-resistant epilepsy. In some cases, folding defects are amenable to rescue by small molecules, known as pharmacological and chemical chaperones, which restore the cell surface expression and transport activity of the previously non-functional proteins. Insights from the recent molecular, animal and human case studies of CTD add toward our understanding of this complex disorder and reveal the wide-ranging effects elicited upon CRT1 dysfunction. This grants novel therapeutic prospects for the treatment of patients afflicted with CTD, e.g., modifying the creatine molecule to facilitate CRT1-independent entry into brain cells, or correcting folding-deficient and loss-of-function CTD variants using pharmacochaperones and/or allosteric modulators. The latter justifies a search for additional compounds with a capacity to correct mutation-specific defects.
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http://dx.doi.org/10.3389/fnsyn.2020.588954DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7644880PMC
October 2020

Functional Impact of the G279S Substitution in the Adenosine A-Receptor (AR-G279S), a Mutation Associated with Parkinson's Disease.

Mol Pharmacol 2020 09;98(3):250-266

Institute of Pharmacology and the Gaston H. Glock Research Laboratories for Exploratory Drug Development, Center of Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria

In medium-size, spiny striatal neurons of the direct pathway, dopamine D- and adenosine A-receptors are coexpressed and are mutually antagonistic. Recently, a mutation in the gene encoding the A-receptor (AR), AR-G279S, was identified in an Iranian family: two affected offspring suffered from early-onset l-DOPA-responsive Parkinson's disease. The link between the mutation and the phenotype is unclear. Here, we explored the functional consequence of the G279S substitution on the activity of the A-receptor after heterologous expression in HEK293 cells. The mutation did not affect surface expression and ligand binding but changed the susceptibility to heat denaturation: the thermodynamic stability of AR-G279S was enhanced by about 2 and 8 K when compared with wild-type A-receptor and AR-Y288A (a folding-deficient variant used as a reference), respectively. In contrast, the kinetic stability was reduced, indicating a lower energy barrier for conformational transitions in AR-G279S (73 ± 23 kJ/mol) than in wild-type AR (135 ± 4 kJ/mol) or in AR-Y288A (184 ± 24 kJ/mol). Consistent with this lower energy barrier, AR-G279S was more effective in promoting guanine nucleotide exchange than wild-type AR. We detected similar levels of complexes formed between D-receptors and wild-type AR or AR-G279S by coimmunoprecipitation and bioluminescence resonance energy transfer. However, lower concentrations of agonist were required for half-maximum inhibition of dopamine-induced cAMP accumulation in cells coexpressing D-receptor and AR-G279S than in those coexpressing wild-type AR. These observations predict enhanced inhibition of dopaminergic signaling by AR-G279S in vivo, consistent with a pathogenic role in Parkinson's disease. SIGNIFICANCE STATEMENT: Parkinson's disease is caused by a loss of dopaminergic input from the substantia nigra to the caudate nucleus and the putamen. Activation of the adenosine A-receptor antagonizes responses elicited by dopamine D-receptor. We show that this activity is more pronounced in a mutant version of the A-receptor (AR-G279S), which was identified in individuals suffering from early-onset Parkinson's disease.
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http://dx.doi.org/10.1124/molpharm.120.000003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116137PMC
September 2020

Allosterically Linked Binding Sites in Serotonin Transporter Revealed by Single Molecule Force Spectroscopy.

Front Mol Biosci 2020 3;7:99. Epub 2020 Jun 3.

Institute of Biophysics, Johannes Kepler University Linz, Linz, Austria.

Crystal structures and experiments relying on the tools of molecular pharmacology reported conflicting results on ligand binding sites in neurotransmitter/sodium symporters (NSS). We explored the number and functionality of ligand binding sites of NSS in a physiological setting by designing novel tools for atomic force microscopy (AFM). These allow for directly measuring the interaction forces between the serotonin transporter (SERT) and the antidepressant S-citalopram (S-CIT) on the single molecule level: the AFM cantilever tips were functionalized with S-CIT via a flexible polyethylene glycol (PEG) linker. The tip chemistry was validated by specific force measurements and recognition imaging on CHO cells. Two distinct populations of characteristic binding strengths of S-CIT binding to SERT were revealed in Na-containing buffer. In contrast, in Li-containing buffer, SERT showed only low force interactions. Conversely, the vestibular mutant SERT-G402H merely displayed the high force population. These observations provide physical evidence for the existence of two binding sites in SERT. The dissociation rate constant of both binding sites was extracted by varying the dynamics of the force-probing experiments. Competition experiments revealed that the two sites are allosterically coupled and exert reciprocal modulation.
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http://dx.doi.org/10.3389/fmolb.2020.00099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7325972PMC
June 2020

Against all odds-the persistent popularity of homeopathy.

Wien Klin Wochenschr 2020 May 9;132(9-10):232-242. Epub 2020 Mar 9.

Association of Medical Students, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria.

The use of homeopathy is remarkably popular. Popularity, however, is not an arbiter in a scientific discourse. In fact, the assumptions underlying homeopathy violate fundamental laws of nature. Homeopathy does not have any explanatory power and fails other criteria established for a scientific approach. Two large-scale efforts have recently documented that in spite of a plethora of clinical trials there is no evidence that homeopathic remedies have any therapeutic effect, which goes beyond that of a placebo. Relaxed regulations and lack of scientific literacy and of health education allow for continuous thriving of homeopathy. While the tide may be changing on the regulatory side, health education of the general public is presumably more important to support informed decision making by patients. Otherwise, the responsible patient, who is posited to decide on the medical choices, remains a convenient legal fiction.
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http://dx.doi.org/10.1007/s00508-020-01624-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7253376PMC
May 2020

Regimen-dependent synergism and antagonism of treprostinil and vildagliptin in hematopoietic cell transplantation.

J Mol Med (Berl) 2020 02 24;98(2):233-243. Epub 2019 Dec 24.

Institute of Pharmacology and the Gaston H. Glock Research Laboratories for Exploratory Drug Development, Medical University of Vienna, 1090, Vienna, Austria.

The cell dose in umbilical cord blood units is a major determinant for the outcome of hematopoietic cell transplantation. Prostaglandin analogs and dipeptidylpeptidase-4 (DPP4/CD26)-inhibitors enhance the ability of hematopoietic stem cells (HSCs) to reconstitute hematopoiesis. Here we explored the synergism between treprostinil, a stable prostaglandin agonist, and the DPP4/CD26-inhibitor vildagliptin. The combination of treprostinil and forskolin caused a modest but statistically significant increase in the surface levels of DPP4/CD26 on hematopoietic stem and progenitor cells (HSPCs) derived from murine bone and human cord blood. Their migration towards stromal cell-derived factor-1 (SDF-1/CXCL12) was enhanced, if they were pretreated with treprostinil and forskolin, and further augmented by vildagliptin. Administration of vildagliptin rescued 25% of lethally irradiated recipient mice injected with a limiting number of untreated HSPCs, but 90 to 100% of recipients injected with HSPCs preincubated with treprostinil and forskolin. The efficacy of vildagliptin surpassed that of treprostinil (60% rescue). Surprisingly, concomitant administration of vildagliptin and treprostinil resulted in poor survival of recipients indicating mutual antagonism, which was recapitulated when homing of and colony formation by HSPCs were assessed. These observations of regimen-dependent synergism and antagonism of treprostinil and vildagliptin are of translational relevance for the design of clinical trials. KEY MESSAGES: Pretreatment with treprostinil increases surface levels of DPP4/CD26 in HSPCs. Vildagliptin enhances in vitro migration of pretreated HSPCs. Vildagliptin enhances in vivo homing and engraftment of pretreated HSPCs. Unexpected mutual antagonism in vivo by concomitant administration of vildagliptin and treprostinil.
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http://dx.doi.org/10.1007/s00109-019-01869-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7007891PMC
February 2020

Effect of rain on absorption after transdermal application of flunixin in calves.

J Vet Pharmacol Ther 2020 Jan 13;43(1):87-90. Epub 2019 Dec 13.

Clinic for Ruminants, University for Veterinary Medicine Vienna, Vienna, Austria.

Flunixin is a nonsteroidal anti-inflammatory drug (NSAID) that has anti-inflammatory, anti-pyretic, and analgesic effects. Recently, a novel transdermal formulation was developed (Finadyne Transdermal, MSD Animal Health) and is now the first NSAID registered to be administered as a pour-on product in cattle. According to the manufacturer's instructions, the pour-on product should be applied only to dry skin and exposure to rain should be avoided for at least 6 hr after application. The objective of the study was to evaluate the effect of simulated exposure to light or heavy rain on flunixin absorption and bioavailability within the first 4 hr after administration. Therefore, an isocratic HPLC method was developed to quantify flunixin concentrations in bovine serum by UV detection. Light rain decreased flunixin absorption only when rain started immediately after flunixin administration, while light rain starting more than 30 min after administration of flunixin had no effect on absorption. Absorption and bioavailability of flunixin was impacted under simulated heavy rain conditions, when exposure to rain occurred within one hour after the application of the pour-on formulation, but not later.
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http://dx.doi.org/10.1111/jvp.12829DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7003742PMC
January 2020

Kinetic Models of Secondary Active Transporters.

Int J Mol Sci 2019 Oct 28;20(21). Epub 2019 Oct 28.

Institute of Pharmacology and the Gaston H. Glock Research Laboratories for Exploratory Drug Development, Center of Physiology and Pharmacology, Medical University of Vienna, 1090 Vienna, Austria.

Kinetic models have been employed to understand the logic of substrate transport through transporters of the Solute Carrier (SLC) family. All SLC transporters operate according to the alternate access model, which posits that substrate transport occurs in a closed loop of partial reactions (i.e., a transport cycle). Kinetic models can help to find realistic estimates for conformational transitions between individual states of the transport cycle. When constrained by experimental results, kinetic models can faithfully describe the function of a candidate transporter at a pre-steady state. In addition, we show that kinetic models can accurately predict the intra- and extracellular substrate concentrations maintained by the transporter at a steady state, even under the premise of loose coupling between the electrochemical gradient of the driving ion and of the substrate. We define the criteria for the design of a credible kinetic model of the SLC transporter. Parsimony is the guiding principle of kinetic modeling. We argue, however, that the level of acceptable parsimony is limited by the need to account for the substrate gradient established by a secondary active transporter, and for random order binding of co-substrates and substrate. Random order binding has consistently been observed in transporters of the SLC group.
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http://dx.doi.org/10.3390/ijms20215365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6862442PMC
October 2019

Optimization of an Antibody Light Chain Framework Enhances Expression, Biophysical Properties and Pharmacokinetics.

Antibodies (Basel) 2019 Sep 6;8(3). Epub 2019 Sep 6.

Baxalta Innovations GmbH (part of Takeda), Indusriestrasse 67, 1221 Vienna, Austria.

Efficacy, safety, and manufacturability of therapeutic antibodies are influenced by their biopharmaceutical and biophysical properties. These properties can be optimized by library approaches or rationale protein design. Here, we employed a protein engineering approach to modify the variable domain of the light chain (VL) framework of an oxidized macrophage migration inhibitory factor (oxMIF)-specific antibody. The amendment of the antibody sequence was based on homology to human germline VL genes. Three regions or positions were identified in the VL domain-L1-4, L66, L79-and mutated independently or in combination to match the closest germline V gene. None of the mutations altered oxMIF specificity or affinity, but some variants improved thermal stability, aggregation propensity, and resulted in up to five-fold higher expression. Importantly, the improved biopharmaceutical properties translated into a superior pharmacokinetic profile of the antibody. Thus, optimization of the V domain framework can ameliorate the biophysical qualities of a therapeutic antibody candidate, and as result its manufacturability, and also has the potential to improve pharmacokinetics.
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http://dx.doi.org/10.3390/antib8030046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6784111PMC
September 2019

An Electrophysiological Approach to Measure Changes in the Membrane Surface Potential in Real Time.

Biophys J 2020 02 5;118(4):813-825. Epub 2019 Jul 5.

Institute of Pharmacology and the Gaston H. Glock Research Laboratories for Exploratory Drug Development, Center of Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria. Electronic address:

Biological membranes carry fixed charges at their surfaces. These arise primarily from phospholipid headgroups. In addition, membrane proteins contribute to the surface potential with their charged residues. Membrane lipids are asymmetrically distributed. Because of this asymmetry, the net-negative charge at the inner leaflet exceeds that at the outer leaflet. Changes in surface potential are predicted to give rise to apparent changes in membrane capacitance. Here, we show that it is possible to detect changes in surface potential by an electrophysiological approach; the analysis of cellular currents relies on assuming that the electrical properties of a cell are faithfully described by a three-element circuit (i.e., the minimal equivalent circuit) comprised of two resistors and one capacitor. However, to account for changes in surface potential, it is necessary to add a battery to this circuit connected in series with the capacitor. This extended circuit model predicts that the current response to a square-wave voltage pulse harbors information, which allows for separating the changes in surface potential from a true capacitance change. We interrogated our model by investigating changes in the capacitance induced by ligand binding to the serotonin transporter and to the glycine transporters (GlyT1 and GlyT2). The experimental observations were consistent with the predictions of the extended circuit. We conclude that ligand-induced changes in surface potential (reflecting the binding event) and in true membrane capacitance (reflecting the concomitant conformational change) can be detected in real time even in instances in which they occur simultaneously.
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http://dx.doi.org/10.1016/j.bpj.2019.06.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7036697PMC
February 2020

A comparison of the transport kinetics of glycine transporter 1 and glycine transporter 2.

J Gen Physiol 2019 08 3;151(8):1035-1050. Epub 2019 Jul 3.

Institute of Pharmacology and the Gaston H. Glock Research Laboratories for Exploratory Drug Development, Center of Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria

Transporters of the solute carrier 6 (SLC6) family translocate their cognate substrate together with Na and Cl Detailed kinetic models exist for the transporters of GABA (GAT1/SLC6A1) and the monoamines dopamine (DAT/SLC6A3) and serotonin (SERT/SLC6A4). Here, we posited that the transport cycle of individual SLC6 transporters reflects the physiological requirements they operate under. We tested this hypothesis by analyzing the transport cycle of glycine transporter 1 (GlyT1/SLC6A9) and glycine transporter 2 (GlyT2/SLC6A5). GlyT2 is the only SLC6 family member known to translocate glycine, Na, and Cl in a 1:3:1 stoichiometry. We analyzed partial reactions in real time by electrophysiological recordings. Contrary to monoamine transporters, both GlyTs were found to have a high transport capacity driven by rapid return of the empty transporter after release of Cl on the intracellular side. Rapid cycling of both GlyTs was further supported by highly cooperative binding of cosubstrate ions and substrate such that their forward transport mode was maintained even under conditions of elevated intracellular Na or Cl The most important differences in the transport cycle of GlyT1 and GlyT2 arose from the kinetics of charge movement and the resulting voltage-dependent rate-limiting reactions: the kinetics of GlyT1 were governed by transition of the substrate-bound transporter from outward- to inward-facing conformations, whereas the kinetics of GlyT2 were governed by Na binding (or a related conformational change). Kinetic modeling showed that the kinetics of GlyT1 are ideally suited for supplying the extracellular glycine levels required for NMDA receptor activation.
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http://dx.doi.org/10.1085/jgp.201912318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6683666PMC
August 2019

How to rescue misfolded SERT, DAT and NET: targeting conformational intermediates with atypical inhibitors and partial releasers.

Biochem Soc Trans 2019 06 7;47(3):861-874. Epub 2019 May 7.

Institute of Pharmacology and the Gaston H. Glock Research Laboratories for Exploratory Drug Development, Center of Physiology and Pharmacology, Medical University of Vienna, Vienna, Waehringerstrasse 13a, Vienna, Austria

Point mutations in the coding sequence for solute carrier 6 (SLC6) family members result in clinically relevant disorders, which are often accounted for by a loss-of-function phenotype. In many instances, the mutated transporter is not delivered to the cell surface because it is retained in the endoplasmic reticulum (ER). The underlying defect is improper folding of the transporter and is the case for many of the known dopamine transporter mutants. The monoamine transporters, i.e. the transporters for norepinephrine (NET/SLC6A2), dopamine (DAT/SLC6A3) and serotonin (SERT/SLC6A4), have a rich pharmacology; hence, their folding-deficient mutants lend themselves to explore the concept of pharmacological chaperoning. Pharmacochaperones are small molecules, which bind to folding intermediates with exquisite specificity and scaffold them to a folded state, which is exported from the ER and delivered to the cell surface. Pharmacochaperoning of mutant monoamine transporters, however, is not straightforward: ionic conditions within the ER are not conducive to binding of most typical monoamine transporter ligands. A collection of compounds exists, which are classified as atypical ligands because they trap monoamine transporters in unique conformational states. The atypical binding mode of some DAT inhibitors has been linked to their anti-addictive action. Here, we propose that atypical ligands and also compounds recently classified as partial releasers can serve as pharmacochaperones.
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http://dx.doi.org/10.1042/BST20180512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6599159PMC
June 2019

para-Trifluoromethyl-methcathinone is an allosteric modulator of the serotonin transporter.

Neuropharmacology 2019 12 24;161:107615. Epub 2019 Apr 24.

Institute of Pharmacology, Medical University, Vienna, Austria. Electronic address:

The transporters for dopamine (DAT) and serotonin (SERT) are important targets in the treatment of psychiatric disorders including major depression, anxiety and attention-deficit hyperactivity disorder. Drugs acting at these transporters can act as inhibitors or as releasers. In addition, it has been recently appreciated that some compounds are less efficacious releasers than amphetamine. Thus, they are classified as partial releasers. Compounds can act on both SERT and DAT or display exquisite selectivity for either SERT or DAT, but the structural basis for selectivity is poorly understood. The trifluoromethyl-substitution of methcathinone in the para-position has been shown to dramatically shift the selectivity of methcathinone (MCAT) towards SERT. Here, we examined MCAT, para-trifluoromethyl-methcathinone (pCFMCAT) and other analogues to understand (i) the determinants of selectivity and (ii) the effects of the para-CF-substitution of MCAT on the transport cycle. We systematically tested different para-substituted MCATs by biochemical, computational and electrophysiological approaches: addition of the pCFgroup, but not of other substituents with larger van der Waal's volume, lipophilicity or polarity, converted the DAT-selective MCAT into a SERT-selective partial releaser. Electrophysiological and superfusion experiments, together with kinetic modelling, showed that pCFMCAT, but not MCAT, trapped a fraction of SERTs in an inactive state by occupying the S2-site. These findings define a new mechanism of action for partial releasers, which is distinct from the other two known binding modes underlying partial release. Our observations highlight the fact that the substrate permeation pathway of monoamine transporters supports multiple binding modes, which can be exploited for drug design. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.
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http://dx.doi.org/10.1016/j.neuropharm.2019.04.021DOI Listing
December 2019

Nature-Derived Peptides: A Growing Niche for GPCR Ligand Discovery.

Trends Pharmacol Sci 2019 05 5;40(5):309-326. Epub 2019 Apr 5.

Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Austria. Electronic address:

G protein-coupled receptors (GPCRs) represent important drug targets, as they regulate pivotal physiological processes and they have proved to be readily druggable. Natural products have been and continue to be amongst the most valuable sources for drug discovery and development. Here, we surveyed small molecules and (poly-)peptides derived from plants, animals, fungi, and bacteria, which modulate GPCR signaling. Among naturally occurring compounds, peptides from plants, cone-snails, snakes, spiders, scorpions, fungi, and bacteria are of particular interest as lead compounds for the development of GPCR ligands, since they cover a chemical space, which differs from that of synthetic small molecules. Peptides, however, face challenges, some of which can be overcome by studying plant-derived compounds. We argue here that the opportunities outweigh the challenges.
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http://dx.doi.org/10.1016/j.tips.2019.03.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6624132PMC
May 2019

Rescue by 4-phenylbutyrate of several misfolded creatine transporter-1 variants linked to the creatine transporter deficiency syndrome.

Neuropharmacology 2019 12 15;161:107572. Epub 2019 Mar 15.

Institute of Pharmacology and the Gaston H. Glock Research Laboratories for Exploratory Drug Development, Center of Physiology and Pharmacology, Medical University of Vienna, Austria. Electronic address:

Diseases arising from misfolding of SLC6 transporters have been reported over recent years, e.g. folding-deficient mutants of the dopamine transporter and of the glycine transporter-2 cause infantile/juvenile Parkinsonism dystonia and hyperekplexia, respectively. Mutations in the coding sequence of the human creatine transporter-1 (hCRT-1/SLC6A8) gene result in a creatine transporter deficiency syndrome, which varies in its clinical manifestation from epilepsy, mental retardation, autism, development delay and motor dysfunction to gastrointestinal symptoms. Some of the mutations in hCRT-1 occur at residues, which are highly conserved across the SLC6 family. Here, we examined 16 clinically relevant hCRT-1 variants to verify the conjecture that they were misfolded and that this folding defect was amenable to correction. Confocal microscopy imaging revealed that the heterologously expressed YFP-tagged mutant CRTs were trapped in the endoplasmic reticulum (ER), co-localised with the ER-resident chaperone calnexin. In contrast, the wild type hCRT-1 reached the plasma membrane. Preincubation of transiently transfected HEK293 cells with the chemical chaperone 4-phenylbutyrate (4-PBA) restored ER export and surface expression of as well as substrate uptake by several folding-deficient CRT-1 mutants. A representative mutant (hCRT-1-P544L) was expressed in rat primary hippocampal neurons to verify pharmacochaperoning in a target cell: 4-PBA promoted the delivery of hCRT-1-P544L to the neurite extensions. These observations show that several folding-deficient hCRT-1 mutants can be rescued. This proof-of-principle justifies the search for additional pharmacochaperones to restore folding of 4PBA-unresponsive hCRT-1 mutants. Finally, 4-PBA is an approved drug in paediatric use: this provides a rationale for translating the current insights into clinical trials. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.
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http://dx.doi.org/10.1016/j.neuropharm.2019.03.015DOI Listing
December 2019

Distinct contribution of axonal and somatodendritic serotonin transporters in drosophila olfaction.

Neuropharmacology 2019 12 6;161:107564. Epub 2019 Mar 6.

Institute of Pharmacology and the Gaston H. Glock Research Laboratories for Exploratory Drug Development, Center of Physiology and Pharmacology, Medical University of Vienna, A-1090 Vienna, Austria. Electronic address:

The serotonin transporter (SERT) regulates serotonergic neurotransmission by retrieving released serotonin and replenishing vesicular stores. SERT is not only delivered to axons but it is also present on the neuronal soma and on dendrites. It has not been possible to restrict the distribution of SERT without affecting transporter function. Hence, the physiological role of somatodendritic SERT remains enigmatic. The SERT C-terminus harbors a conserved RI-motif, which recruits SEC24C, a cargo receptor in the coatomer protein-II coat. Failure to engage SEC24C precludes axonal enrichment of SERT. Here we introduced a point mutation into the RI-motif of human SERT causing confinement of the resulting - otherwise fully functional - hSERT-RA on the somatodendritic membrane of primary rat dorsal raphe neurons. Transgenic expression of the corresponding Drosophila mutant dSERT-RA led to its enrichment in the somatodendritic compartment of serotonergic neurons in the fly brain. We explored the possible physiological role of somatodendritic SERT by comparing flies harboring wild type SERT and dSERT-RA in a behavioral paradigm for serotonin-modulated odor perception. When globally re-expressed in serotonergic neurons, wild type SERT but not dSERT-R599A restored ethanol preference. In contrast, dSERT-R599A restored ethanol preference after targeted expression in contralaterally projecting, serotonin-immunoreactive deuterocerebral (CSD) interneurons, while expression of wild type SERT caused ethanol aversion. We conclude that, in CSD neurons, (i) somatodendritic SERT supports ethanol attraction, (ii) axonal SERT specifies ethanol aversion, (iii) the effect of axonal SERT can override that of somatodendritic SERT. These observations demonstrate a distinct biological role of somatodendritic and axonal serotonin transport. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.
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http://dx.doi.org/10.1016/j.neuropharm.2019.03.007DOI Listing
December 2019

Functional Selectivity and Partial Efficacy at the Monoamine Transporters: A Unified Model of Allosteric Modulation and Amphetamine-Induced Substrate Release.

Mol Pharmacol 2019 03 19;95(3):303-312. Epub 2018 Dec 19.

Institute of Pharmacology, Gaston H. Glock Research Laboratories for Exploratory Drug Development, Center of Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria.

All clinically approved drugs targeting the plasmalemmal transporters for dopamine, norepinephrine, and serotonin act either as competitive uptake inhibitors or as amphetamine-like releasers. Monoamine transporter (MAT) ligands that allosterically affect MAT-mediated substrate uptake, release, or both were recently discovered. Their modes of action have not yet been explained in a unified framework. Here, we go beyond competitive inhibitors and classic amphetamines and introduce concepts for partial efficacy at and allosteric modulation of MATs. After we elaborate on a kinetic account for amphetamine action, we provide an explanation for partial release (i.e., the observation that some amphetamines are less efficacious than others in inducing monoamine efflux). We then elucidate mechanisms of allosteric inhibition and stimulation of MATs, which can be functionally selective for either substrate uptake or amphetamine-induced release. These concepts are integrated into a parsimonious kinetic framework, which relies exclusively on physiologic transport modes (without any deviation from an alternating access mechanism). The model posits cooperative substrate and Na binding and functional selectivity by conformational selection (i.e., preference of the allosteric modulators for the substrate-loaded or substrate-free states of the transporter). Thus, current knowledge about the kinetics of monoamine transport is sufficiently detailed to provide a quantitative description of the releasing action of amphetamines, of substrate uptake, and of selective modulation thereof by allosteric modulators.
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http://dx.doi.org/10.1124/mol.118.114793DOI Listing
March 2019

Treprostinil reduces endothelial damage in murine sinusoidal obstruction syndrome.

J Mol Med (Berl) 2019 02 7;97(2):201-213. Epub 2018 Dec 7.

Institute of Pharmacology and the Gaston H. Glock Research Laboratories for Exploratory Drug Development, Centre of Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria.

Sinusoidal obstruction syndrome (SOS) is a major complication after hematopoietic stem cell transplantation and belongs to a group of diseases increasingly identified as transplant-related systemic endothelial disease. Administration of defibrotide affords some protection against SOS, but the effect is modest. Hence, there is unmet medical need justifying the preclinical search for alternative approaches. Prostaglandins exert protective actions on endothelial cells of various vascular beds. Here, we explored the therapeutic potential of the prostacyclin analog treprostinil to prevent SOS. Treprostinil acts via stimulation of IP, EP, and EP receptors, which we detected in murine liver sinusoidal endothelial cells (LSECs). Busulfan-induced cell death was reduced when pretreated with treprostinil in vitro. In a murine in vivo model of SOS, concomitantly administered treprostinil caused lower liver weight-to-body weight ratios indicating liver protection. Histopathological changes were scored to assess damage to liver sinusoidal endothelial cells, to hepatocytes, and to the incipient fibrotic reaction. Treprostinil indeed reduced sinusoidal endothelial cell injury, but this did not translate into reduced liver cell necrosis or fibrosis. In summary, our observations provide evidence for a beneficial effect of treprostinil on damage to LSECs but unexpectedly treprostinil was revealed as a double-edged sword in SOS. KEY MESSAGES: Murine liver sinusoidal endothelial cells (LSECs) express prostanoid receptors. Treprostinil reduces busulfan-induced cell death in vitro. Treprostinil lowers liver weight-to-body weight ratios in mice. Treprostinil positively affects LSECs in mice but not hepatic necrosis/fibrosis.
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http://dx.doi.org/10.1007/s00109-018-1726-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6348071PMC
February 2019
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