Papers by Michael Freissmuth
PLOS ONE, May 27, 2021
Clostridioides difficile (C. difficile) infection is a major public health problem worldwide. The... more 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.
European Journal of Pharmacology
The antidepressant-like activity of (+)-catharanthine and (-)-18-methoxycoronaridine [(-)-18-MC] ... more The antidepressant-like activity of (+)-catharanthine and (-)-18-methoxycoronaridine [(-)-18-MC] was studied in male and female mice using forced swim (FST) and tail suspension tests (TST). The underlying molecular mechanism was assessed by electrophysiological, radioligand, and functional experiments. The FST results showed that acute administration (40 mg/kg) of (+)-catharanthine or (-)-18-MC induces similar antidepressant-like activity in male and female mice at 1 h and 24 h, whereas the TST results showed a lower effect for (-)-18-MC at 24 h. Repeated treatment at lower dose (20 mg/kg) augmented the efficacy of both congeners. The FST results showed that (-)-18-MC reduces immobility and increases swimming times without changing climbing behavior, whereas (+)-catharanthine reduces immobility time, increases swimming times more markedly, and increases climbing behavior. To investigate the contribution of the serotonin and norepinephrine transporters in the antidepressant effects of (+)catharanthine and (-)-18-MC, we conducted in vitro radioligand and functional studies. Results obtained demonstrated that (+)-catharanthine inhibits norepinephrine transporter with higher potency/affinity than that for (-)-18-MC, whereas both congeners inhibit serotonin transporter with similar potency/affinity. Moreover, whereas no congener activated/inhibited/potentiated the function of serotonin receptor 3A or serotonin receptor 3AB, both increased serotonin receptor 3A receptor desensitization. Depletion of serotonin decreased the antidepressant-like activity of both congeners, whereas norepinephrine depletion only decreased (+)-catharanthine's activity. Our study shows that coronaridine congeners induce antidepressant-like activity in a dose-and time-dependent, and sex-independent, manner. The antidepressant-like property of both compounds involves serotonin transporter inhibition, without directly activating/inhibiting serotonin receptors 3, while (+)-catharanthine also mobilizes norepinephrinergic neurotransmission.
Amphetamines and cocaine are illicitly used psychostimulants which act on monoaminergic neurotran... more Amphetamines and cocaine are illicitly used psychostimulants which act on monoaminergic neurotransmitter transporters. These comprise the transporters for dopamine, serotonin, or norepinephrine. By themselves, the drugs can already cause severe problems in terms of neurotoxicity and addiction. Nevertheless, additional health threats arise from the possibility that these compounds are cut by adding adulterant substances without declaration. This chapter examines commonly found adulterants of drugs which are sold as cocaine on the street market. The typical pharmacological profile is discussed and the chapter elaborates on reasons why these compounds can be used as cutting agents. It has been found that a subset of these adulterants exerts effects similar to cocaine itself. Furthermore, we will discuss the example of levamisole, which is the most frequently used cocaine cutting agent today, and its metabolite aminorex.
Cells
Organic cation transporters (OCTs) are membrane proteins that take up monoamines, cationic drugs ... more Organic cation transporters (OCTs) are membrane proteins that take up monoamines, cationic drugs and xenobiotics. We previously reported novel missense mutations of organic cation transporter 3 (OCT3, SLC22A3), some with drastically impacted transport capabilities compared to wildtype. For some variants, this was due to ER retention and subsequent degradation of the misfolded transporter. For other transporter families, it was previously shown that treatment of misfolded variants with pharmacological and chemical chaperones could restore transport function to a certain degree. To investigate two potentially ER-bound, misfolded variants (D340G and R348W), we employed confocal and biochemical analyses. In addition, radiotracer uptake assays were conducted to assess whether pre-treatment with chaperones could restore transporter function. We show that pre-treatment of cells with the chemical chaperone 4-PBA (4-phenyl butyric acid) leads to increased membrane expression of misfolded var...
The serotonin transporter (SERT/SLC6A4) is arguably the most extensively studied solute carrier (... more The serotonin transporter (SERT/SLC6A4) is arguably the most extensively studied solute carrier (SLC). During its eponymous action - i.e., the retrieval of serotonin from the extracellular space - SERT undergoes a conformational cycle. Typical inhibitors (antidepressant drugs and cocaine), partial and full substrates (amphetamines and their derivatives) and atypical inhibitors (ibogaine analogues) bind preferentially to different states in this cycle. This results in competitive or non-competitive transport inhibition. Here, we explored the action of N-formyl-1,3-bis (3,4-methylenedioxyphenyl)-prop-2-yl-amine (ECSI#6) on SERT: inhibition of serotonin uptake by ECSI#6 was enhanced with increasing serotonin concentration. Conversely, the KM for serotonin was lowered by augmenting ECSI#6. ECSI#6 bound with low affinity to the outward-facing state of SERT but with increased affinity to a potassium-bound state. Electrophysiological recordings showed that ECSI#6 preferentially interacted ...
Frontiers in Pharmacology, 2021
Objectives: Reimbursement decisions on new medicines require an assessment of their value. In Aus... more Objectives: 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. Methods: 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. Results: 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 b...
eLife, 2021
The concentrative power of the transporters for dopamine (DAT), norepinephrine (NET), and seroton... more 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...
The concentrative power of the transporters for dopamine (DAT), norepinephrine (NET), and seroton... more 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 voltagedependent 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.
Scientific Reports, 2021
Lead exposure can cause substantial organ damage. Enteral lead absorption may be reduced by conco... more 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 204Pb 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 204Pb in water. The enrichment of 204Pb caused by the tracer in blood and urine was measured by mass spectrometry. G-PUR was well tolerated. The mean maximum 204Pb 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 204Pb AUC0-192 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)....
Frontiers in Pharmacology, 2021
Over the past years, peptides have attracted increasing interest for G protein-coupled receptor (... more Over the past years, peptides have attracted increasing interest for G protein-coupled receptor (GPCR) drug discovery and development. Peptides occupy a unique chemical space that is not easily accessible for small molecules and antibodies and provide advantages over these ligand classes such as lower toxicity and higher selectivity. The κ-opioid receptor (KOR) is a prototypic GPCR and an appealing therapeutic target for the development of safer and more effective analgesics. Recently, peptides have emerged as analgesic drug candidates with improved side effect profiles. We have previously identified plant-derived peptides, which activate KOR. Based on this precedent, here we relied on publicly available databases to discover novel KOR peptide ligands by genome mining. Using human preprodynorphin as a query, we identified blenny fish-derived peptides, referred to as blenniorphins, capable of binding to and activating KOR with nanomolar affinity and potency, respectively. Additionall...
Science Translational Medicine, 2021
Viral vectors restore dopamine transporter function and ameliorate neuropathology in iPSC-derived... more Viral vectors restore dopamine transporter function and ameliorate neuropathology in iPSC-derived neurons and a mouse model of infantile parkinsonism.
Journal of Biological Chemistry, 1994
We have searched for irreversible ligands which target the guanine nucleotide binding pocket of G... more We have searched for irreversible ligands which target the guanine nucleotide binding pocket of G protein alpha-subunits by testing the ability of periodate-oxidized 2',3'-dialdehyde guanine nucleotide analogues of GTP (oGTP) and GTP gamma S (oGTP gamma S) to bind to the recombinant alpha-subunit of the stimulatory G protein, rGs alpha-s. oGTP and oGTP gamma S bind to rGs alpha-s in a quasi-irreversible manner via formation of a Schiff's base, which can be reduced with borhydrid resulting in covalent incorporation of [alpha-32P]oGTP and [35S]oGTP gamma S into rGs alpha-s. When bound to rGs alpha-s, oGTP is hydrolyzed and traps the protein in the inactive conformation, while oGTP gamma S persistently activates rGs alpha. Thus, oGTP and oGTP gamma S act as irreversible G protein antagonist and agonist, respectively, and represent a pair of nucleotide analogues suitable as functional and structural tools. Cleavage of covalently labeled rGs alpha-s with cyanogen bromide generates several labeled fragments. Labeled fragments were assigned to the G1 and G4 region of the guanine nucleotide binding pocket using sequence-specific antisera. An additional, labeled fragment was identified by amino-terminal sequencing and corresponded to the helix alpha A in the recently determined crystal structure of the transducin alpha-subunit (Noel, J. P., Hamm, H. E., and Sigler, P. B. (1993) Nature 366, 654-663). In the oGDP-liganded conformation, incorporation occurs predominantly into the G1-fragment, while [35S]oGTP gamma S labels the additional fragments to a similar extent indicating tight packing around the guanine nucleotide binding pocket in the active conformation. Furthermore, rGs alpha-s contains a single acid cleavable bond (Asp317-Pro318), such that formic acid releases a carboxyl-terminal fragment from [alpha-32P]oGTP- and [35S]oGTP gamma S-liganded rGs alpha-s. This fragment contains a single lysine residue (Lys324) which is only labeled by [35S]oGTP gamma S. Lys324 is unique to Gs alpha and lies within its effector binding region. Hence, during the switch from the inactive to the active state, this region undergoes a major conformational change that moves it closer to the nucleotide binding pocket.
Journal of Biological Chemistry, 1994
Frontiers in Synaptic Neuroscience, 2020
(influenza, neumococo y tétanos) en adultos mayores de 60 años en México.
The dopamine transporter (DAT) retrieves dopamine into presynaptic terminals after synaptic relea... more The dopamine transporter (DAT) retrieves dopamine into presynaptic terminals after synaptic release. The concentrative power of DAT is thought to be fueled by the transmembrane Na+gradient, but it is conceivable that DAT can also rely on other energy sources, e.g. membrane voltage and/or the K+gradient. Here, we recorded uptake of dopamine or the fluorescent substrate APP+((4-(4-dimethylamino)phenyl-1-methylpyridinium) in DAT-expressing cells under voltage control. We show that DAT differs substantially from the closely related serotonin transporter (SERT): substrate uptake by DAT was voltage-dependent, intracellular K+binding to DAT was electrogenic but transient in nature thus precluding antiport of K+by DAT. There is a trade-off between maintaining constant uptake and harvesting membrane potential for concentrative power. Based on our observations, we conclude that subtle differences in the kinetics of co-substrate ion binding allow closely related transporters to select between ...
Journal of Veterinary Pharmacology and Therapeutics, 2019
Flunixin is a nonsteroidal anti‐inflammatory drug (NSAID) that has anti‐inflammatory, anti‐pyreti... more 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 ...
Journal of General Physiology, 2019
Transporters of the solute carrier 6 (SLC6) family translocate their cognate substrate together w... more 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 cosubstra...
Neuropharmacology, 2019
The transporters for dopamine (DAT) and serotonin (SERT) are important targets in the treatment o... more 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 trifluoromethylsubstitution 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 (p-CF 3-MCAT) and other analogues to understand (i) the determinants of selectivity and (ii) the effects of the para-CF 3-substitution of MCAT on the transport cycle. We systematically tested different parasubstituted MCATs by biochemical, computational and electrophysiological approaches: addition of the p-CF 3-group, 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 modeling, showed that p-CF 3-MCAT, 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.
Trends in Pharmacological Sciences, 2019
G protein-coupled receptors (GPCRs) represent important drug targets, as they regulate pivotal ph... more 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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Papers by Michael Freissmuth
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