key: cord-0917469-4rz1297k authors: Krisanova, Natalia; Pozdnyakova, Natalia; Pastukhov, Artem; Dudarenko, Marina; Shatursky, Oleg; Gnatyuk, Olena; Afonina, Uliana; Pyrshev, Kyrylo; Dovbeshko, Galina; SemenYesylevskyy; Borisova, Tatiana title: Amphiphilic anti-SARS-CoV-2 drug remdesivir incorporates into the lipid bilayer and nerve terminal membranes influencing excitatory and inhibitory neurotransmission date: 2022-04-22 journal: Biochim Biophys Acta Biomembr DOI: 10.1016/j.bbamem.2022.183945 sha: c41fd1c82aa1f7a366d20505160cb7f416ad4fbe doc_id: 917469 cord_uid: 4rz1297k Remdesivir is a novel antiviral drug, which is active against the SARS-CoV-2 virus. Remdesivir is known to accumulate in the brain but it is not clear whether it influences the neurotransmission. Here we report diverse and pronounced effects of remdesivir on transportation and release of excitatory and inhibitory neurotransmitters in rat cortex nerve terminals (synaptosomes) in vitro. Direct incorporation of remdesivir molecules into the cellular membranes was shown by FTIR spectroscopy, planar phospholipid bilayer membranes and computational techniques. Remdesivir decreases depolarization-induced exocytotic release of L-[(14)C] glutamate and [(3)H] GABA, and also [(3)H] GABA uptake and extracellular level in synaptosomes in a dose-dependent manner. Fluorimetric studies confirmed remdesivir-induced impairment of exocytosis in nerve terminals and revealed a decrease in synaptic vesicle acidification. Our data suggest that remdesivir dosing during antiviral therapy should be precisely controlled to prevent possible neuromodulatory action at the presynaptic level. Further studies of neurotropic and membranotropic effects of remdesivir are necessary. partial charges were computed, averaged for topologically equivalent atoms and applied to generated topology. The model membrane was generated previously with the lipid composition close to one of the plasma membrane of human lung epithelial cells. We are aware that this composition could be different from synaptosomes, thus obtained results should be interpreted on qualitative level only. We used the average abundance of the lipid head groups from [15, 16] and the distribution of the lipid tails inside each lipid class from [17] . Only lipid tails with more than 5% abundance were taken into account. Asymmetry of the lipid content of monolayers was set by putting all PC, SM and ceramide lipids to the outer leaflet and all PE, PS, PI and plasmalogen lipids to the inner leaflet. Cholesterol was distributed equally between the leaflets. CHARMM GUI membrane builder [12, 13] with the lipid composition detailed in Table 1 was used to compose the membrane patch containing 64 lipids in each monolayer. The membrane was duplicated in X direction to create an elongated membrane patch containing 384 lipids. The system was solvated by ~40000 water molecules. The number of ions corresponds to 0.15 M of NaCl with additional counter ions added to neutralize the system. Additional slabs of water were added in X direction to convert the system into the bicelle, which is periodic in Y direction. The advantage of the bicelle setup is the absence of the lateral strain caused by uncompensated difference in the areas per lipid in inner and outer leaflets. The bicelle caps serve as reservoirs which accumulate excessive lipids from one monolayer and donate them to the other. As a result both monolayers possess an optimal number of lipids, which changes dynamically during the simulation. The mixing of lipids from different monolayers in the bicelle caps was prevented by means of selective repulsive potentials as described elsewhere [18] [19] [20] [21] [22] [23] . The flat form of the bicelle was maintained by means of EnCurv technique [24] with enforced zero curvature. The bicelle was equilibrated for 200 ns. Outer leaflet Inner leaflet DPPC 5 21, 2020) . All animal studies were reported in accordance to the ARRIVE guidelines for reporting experiments involving animals [38, 39] . The total number of rats used in the study was 36 , specifically, the measurements of the extracellular levels and uptake of L-[ 14 The cortex brain region isolated from decapitated rats was rapidly removed, and then homogenized in the ice-cold solution containing 0.32 M sucrose, 5 mM HEPES-NaOH, pH 7.4, and 0.2 mM EDTA. One synaptosome preparation was obtained from one rat, and each measurement was done in triplicate. The synaptosome preparations were obtained using differential and Ficoll-400 density gradient centrifugations of rat brain homogenate according to [40] [41] [42] . and D-glucose 10. Ca 2+ -containing media were supplemented with 2 mM CaCl 2. Ca 2+ -free media were supplemented with 2 mM EGTA . Protein concentration was examined according to [43] . Synaptosomes without remdesivir and after incubation with remdesivir (0.028 mg/ml) were analysed using INVENIO-R instrument (Bruker, Germany). Two types of preparations of synaptosomes of different functional states were used in FTIR experiments. The first one was functionally active synaptosomes with normal membrane potential, which were characterized by a high capability to accumulate potential-sensitive fluorescent dye rhodamine 6 G. The accumulation level was 0.2 a.u., calculated as F = F t /F 0, where F 0 and F t were fluorescence intensities of rhodamine 6 G in the absence and presence of the synaptosomes, respectively. The second one was functionally inert synaptosomes, which were kept in freezer under -24 C o during few weeks. They were characterized by a low capability to accumulate rhodamine 6 G. The accumulation level was less than 0.95 a.u. (no membrane potential), calculated according to above equation. To register the FTIR-ATR spectra, 20 μl of the synaptosome suspension was applied to ZnSe crystal of the Bio-ATR attachment. The sample was dried in the spectrometer sample compartment for 1 hour in a stream of nitrogen at room temperature fixing a state of synaptosomes at the present moment. The spectra were registered until the OH band at 3400 cm -1 stopped changing and the sample was dried. For FTIR-ATR spectra, the baseline correction and band intensity normalization by the Amide I band centered at 1652 cm -1 have been done on the J o u r n a l P r e -p r o o f Journal Pre-proof basis of supposition that a number of protein molecules was constant in the synaptosomes of both functional states. The synaptosome preparations were diluted in the standard salt solution to reach a concentration of 2 mg of protein/ml, and after pre-incubation at 37 GABA uptake data were collected in triplicate from several (n) independent experiments performed with different synaptosome preparations. Acridine orange, a pH-sensitive fluorescent dye, was selectively accumulated by the acid compartments of nerve terminals, namely synaptic vesicles [50] . The experimental data were expressed as the mean ± S.E.M. of n independent experiments. The difference between two groups was compared by one-way ANOVA. Differences were considered significant, when p < 0.05. Remdesivir molecules incorporate spontaneously into the membrane during first 10-20 ns and remain intercalated between the lipids of the outer leaflet during the whole simulation. Visual inspection shows no signs of aggregation or dimerization of remdesivir molecules despite its active lateral diffusion. Typical positions and orientations of REM molecules in the model membrane are shown in Fig.1 a. In general, remdesivir sits rather deep in the bilayer, on average ~1.7 nm from its center. It is subject to substantial normal and lateral diffusive motions in the course of simulations but it never detaches from bilayer or changes its preferred orientation. Although the simulations were performed for complex lipid mixture mimicking the composition of the cellular membranes of lung epithelial cells, no specific interactions of remdesivir with particular lipid species were observed. Spontaneous incorporation or remdesivir molecules occurs randomly in respect to exposed lipids and is driven by the minimization of hydrophobic mismatch of its aliphatic moieties and favourable interaction of the polar moieties with the lipid head groups and water. This suggests that the same thermodynamic mechanism, which is similar for the majority of amphiphilic membranotropic compounds, also drives incorporation of remdesivir into the simpler model membranes containing only few lipid species. Our simulations do not allow studying specific influence of lipid unsaturation, polarity and charge on the incorporation of remdesivir. In order to get such information, the series of The introduction of remdesivir at a concentration of 100 M to PC/cholesterol BLM kept at 2:1 weight ratio was followed by the appearance of step-like increases in transmembrane current indicative of pore-formation (Fig 2 a) . This included relatively stable increases in current and those interrupted by brief falls within a fluctuating diameter of each separate step, which suggest rapid resealing of the single pore (Fig 2 a) . The mean unitary conductance of stable single-pore events varied in magnitude within the range of ~10 pS to ~360 pS at a holding potential of 100 mV ( Fig. 2 a, insert). Similar distribution of stable single-pore conductance events was also represented by amplitudes obtained at a negative voltage, -100 mV applied to the same side of membrane except for the appearance of larger increases in current ( Fig. 2 a) . The pore-formation occurred in the symmetrical membrane washing water solution of 100 mM KCl at 100 mV voltage applied on the side of membrane to which remdesivir was added, regardless to the sign of the membrane potential ( Fig. 2 a) . This suggests that the sign of membrane potential is not the necessary prerequisites for remdesivir-induced pore-formation. It Increasing amount of stable, lack in complete resealing pores eventually formed a temporary quasi steady-state integral current consisting of many remdesivir pores (Fig. 2 b) . Conversely, lasting remdesivir-induced increase in current could be partially stabilized by flushing drugcontaining compartment with drug-free saline. This suggests mostly irreversible binding of remdesivir with sterol-containing almost neutral phospholipid bilayer. The effect of applied voltage gradients on magnitude of single pore conductance and a steady-state current flowing across remdesivir-modified bilayer was explored by switching holding potentials between ±100 mV (Figs. 2 a, b). The steady-state macroscopic current J o u r n a l P r e -p r o o f Journal Pre-proof induced by the drug in cholesterol-containing phospholipid bilayer exhibited almost no dependence upon the sign of the membrane potential with a rectification asymmetry coefficient of 0.9 (Fig.2 a) . Linear current-voltage dependence at positive and negative voltages applied to the side opposite to which remdesivir was added also implies that at 2-times lower holding potential (±50 mV), the single pore conductance and summary current will be reduced approximately by a factor of 2 for the type of BLMs tested. The Nernst potential obtained for summary remdesivir-induced current in the membrane washing saline of 10 mM KCl on addition side and 100 mM KCl on the opposite side of PC/cholesterol membrane consisted of 0 mV (Fig. 2 a) . This suggests the lack in cation/anion selectivity of remdesivir-created summary current [51] . Interaction of remdesivir with synaptosomes was examined using FTIR-ATR spectroscopy. of the synaptosome spectra after incubation with remdesivir from the spectra of control synaptosomes without remdesivir). Functionally active synaptosomes were incubated at 37°C for 5 min to restore their ion gradients [44] . To register the FTIR-ATR spectra, 20 μl of the synaptosome suspension was applied to the ZnSe crystal of the Bio-ATR attachment. After that the sample was dried in the spectrometer sample compartment for 1 hour in a stream of nitrogen. The spectra were registered until the OH band at 3400 cm -1 stopped changing and the sample J o u r n a l P r e -p r o o f was dried. After recording the spectra, the baseline was corrected and the absorption spectra were normalized. Functionally inert synaptosomes (Fig. 3 b) showed different changes after remdesivir administration as compared to the functionally active ones (Fig. 3 a) . KCl (35 mM) -induced Ca 2+ -dependent exocytotic release of L-[ 14 C]glutamate from nerve terminals was decreased by remdesivir in a dose-dependent manner (Fig. 4 a) As shown in As shown in Table 2 , remdesivir did not change the initial rate of uptake and accumulation of L-[ 14 C] glutamate for 10 min by nerve terminals. GABA uptake in a dose-dependent manner starting from a concentration of 10 M, thereby demonstrating diverse effects on uptake of excitatory and inhibitory neurotransmitters. Remdesivir (Fig. 5 b) . Therefore, remdesivir did not change the release of L-[ 14 C] glutamate via glutamate transporter reversal, but decreased that of [ 3 H] GABA starting from a concentration of 100 M. The classical protonophore carbonylcyanide p-trifluoromethoxyphenylhydrazone One of the causes of remdesivir-induced mitigation of exocytotic release can be changes in filling of synaptic vesicles with neurotransmitters that depends on the V-ATPase functioning and the proton gradient across the vesicle membrane. A рН-sensitive fluorescent dye acridine orange was applied to measure the synaptic vesicle acidification, as a component of the electrochemical proton gradient [52] . As shown in Figure 5 ***, p < 0.001 as compared to the control; n = 12. To our knowledge current study is the first report of strong interaction of remdesivir with the cellular membrane and its deep incorporation in the lipid bilayer demonstrated by means of molecular dynamics simulations (Fig. 1 a, b) , BLMs technique (Fig. 2 a, b) and FTIR spectroscopy (Fig. 3) . Viral envelopes consist of proteins and lipid components derived from the membranes of host cells [53] . Therefore, it can be hypothesized that remdesivir can incorporate into the viral and membrane active substances and nanoparticles [49, [55] [56] [57] [58] Transporter-mediated L-[ 14 C] glutamate release (Fig. 5 a) was not changed by remdesivir, and so agreed with the abovementioned extracellular level (Fig. 4 с) and uptake data (Table 2) . Whereas, remdesivir differently influenced this parameter in GABA-ergic nerve terminals and decreased transportermediated [ 3 H] GABA release staring from a concentration of 100 M (Fig.5 b) . [ 3 H] GABA data may be explained by decreased surface expression of GABA transporters due to impaired substrate-dependent regulation of GABA transporter functioning via protein kinase C-dependent mechanism in the presence of remdesivir. In this context, a decreased number or changed subtype distribution of functionally active GABA transporters may result in decreased uptake, reduced cytosolic pool of the neurotransmitter, and thus weakened transporter-mediated release, the extracellular level and turnover of neurotransmitter across the plasma membrane [59, 60] . These effects of remdesivir may be due to its capability to form active nucleoside triphosphate [10] that can replace ATP molecule in ATP hydrolysis-dependent synaptic processes and ATP-associated receptor signalling, for instance, exocytosis, functioning of Na-K ATPase, V-ATPase, P2X7 receptors, etc. In particular, capability of remdesivir to interact with V-ATPase was predicted in computational modelling [61] that in turn may result in decreased synaptic vesicle acidification and decreased vesicular neurotransmitter content. Also, elevated extracellular levels of ATP evoked by SARS-CoV-2 infection may trigger hyperactivation of P2X7 receptors, which are ATP-gated ion channels widely expressed in the central nervous system [62] . In particular, P2X7 receptor activation evoked by viral infection led to alterations in reactive oxygen species formation, increased Ca 2+ influx and glutamate release [63, 64] . In turn, glutamate activates NMDA receptors expressed in nerve terminals, which determine Ca 2+dependent exocytosis of ATP and more glutamate release, causing a massive release of the neurotransmitters augmenting excitotoxicity and cell death [1] . It can be suggested that remdesivir-derived nucleoside triphosphate may prevent hyperactivation of P2X7 receptors evoked by SARS-CoV-2, whereas this suggestion needs to be further investigated. A special attention should be payed to the fact that remdesivir and drug-derived material can be accumulated in the brain without changes in their concentrations during 7 days after administration [10] . It is clear that remdesivir concentrations must be precisely controlled during antiviral therapy, because the drug action can be accompanied by dysregulation of synaptic neurotransmission. Indeed, the literature data described usable remdesivir concentrations within the concentration ranges similar to this study. In patient treatment, remdesivir dosage was 200 mg intravenously on day 1 and 100 mg intravenously on day 2-10 [67, 68] . In studies in vitro, active nucleoside triphosphate formation in human monocyte-derived macrophages was analysed following 72-h incubation with 1 μM remdesivir [10] . Remdesivir inhibited acute Ebola virus replication in human cells including primary macrophages and human endothelial cells with halfmaximum effective concentration (EC50) values of 0.06 to 0.14 μM. Our data on changes in glutamate and GABA neurotransmission are in accordance with reported side effects of remdesivir, e.g. nervous system disorders (~ 3 %) and psychiatric disorders (~ 1%) [69] . Identified neuropsychological toxicities of remdesivir were headache, anxiety, seizures, lethargy, delirious symptoms, and poor mental status [68, [70] [71] [72] . Also, hypoxia (~2.5 %) was also shown as side effect of remdesivir [69] that in turn can significantly aggravate remdesivir-induced disturbance in neurotransmitter transport shown in our study. Other most common adverse events of remdesivir in the treatment of critically ill patients were increased hepatic enzymes, diarrhea, rash, renal impairment, hypotension, multiple-organdysfunction syndrome, and septic shock [68] . To date, many studies have been revealed the central nervous system toxicity in response to application of antiviral agents, which may cause severe neuropsychiatric complications, i.e. depression, psychosis, painful peripheral neuropathy, irritability, difficulty sleeping, etc. Neuropsychiatric effects of antiviral drugs are a common occurrence, which complicates treatment [73] . Pathogenetic mechanisms may involve different molecular targets, including GABA A receptors. Peripheral neuropathy for nucleoside and nucleotide analogues is more noticeable with higher dosage and prolonged duration of exposure [73] . Nucleotide analogues used to treat human immunodeficiency virus or hepatitis C virus can cause sensorineural J o u r n a l P r e -p r o o f peripheral neuropathy and other neurological toxicities [74, 75] . It has been suggested that the reaction to most antiviral drugs is idiosyncratic, and the mechanisms of neuropsychiatric effects of these drugs are still unclear, and further research is warranted to elucidate this fact [73] . Drugs used in the palliative treatment of COVID-19 patients also have neurotoxic effects [76] . Antiretroviral drugs targeted coronavirus replication may produce undesirable effects on the central/peripheral nervous systems, variable in frequency and severity, depending on the involved molecular mechanisms [77] . Despite poor penetration through the blood-brain barrier, these drugs are essentially neurotoxic, showing perioral and peripheral paresthesias, and changes in taste within the first month of treatment [78] . In particular, the combination of lopinavir and ritonavir was associated with bilateral sensorineural hearing loss (in 4 weeks of treatment), and the appearance of depressive symptoms. In contrast, darunavir did not show increased neurotoxicity [77, 78] . Direct membranotropic and neurotropic effects of remdesivir are shown for the first time. Remdesivir is able to accumulate in the cell membranes and affects lipid interactions. 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