Dopamine reuptake and inhibitory mechanisms in human dopamine transporter.

The dopamine transporter has a crucial role in regulation of dopaminergic neurotransmission by uptake of dopamine into neurons and contributes to the abuse potential of psychomotor stimulants1-3. Despite decades of study, the structure, substrate binding, conformational transitions and drug-binding poses of human dopamine transporter remain unknown. Here we report structures of the human dopamine transporter in its apo state, and in complex with the substrate dopamine, the attention deficit hyperactivity disorder drug methylphenidate, and the dopamine-uptake inhibitors GBR12909 and benztropine. The dopamine-bound structure in the occluded state precisely illustrates the binding position of dopamine and associated ions. The structures bound to drugs are captured in outward-facing or inward-facing states, illuminating distinct binding modes and conformational transitions during substrate transport. Unlike the outward-facing state, which is stabilized by cocaine, GBR12909 and benztropine stabilize the dopamine transporter in the inward-facing state, revealing previously unseen drug-binding poses and providing insights into how they counteract the effects of cocaine. This study establishes a framework for understanding the functioning of the human dopamine transporter and developing therapeutic interventions for dopamine transporter-related disorders and cocaine addiction.

A regenerative approach to the treatment of multiple sclerosis.

Progressive phases of multiple sclerosis are associated with inhibited differentiation of the progenitor cell population that generates the mature oligodendrocytes required for remyelination and disease remission. To identify selective inducers of oligodendrocyte differentiation, we performed an image-based screen for myelin basic protein (MBP) expression using primary rat optic-nerve-derived progenitor cells. Here we show that among the most effective compounds identifed was benztropine, which significantly decreases clinical severity in the experimental autoimmune encephalomyelitis (EAE) model of relapsing-remitting multiple sclerosis when administered alone or in combination with approved immunosuppressive treatments for multiple sclerosis. Evidence from a cuprizone-induced model of demyelination, in vitro and in vivo T-cell assays and EAE adoptive transfer experiments indicated that the observed efficacy of this drug results directly from an enhancement of remyelination rather than immune suppression. Pharmacological studies indicate that benztropine functions by a mechanism that involves direct antagonism of M1 and/or M3 muscarinic receptors. These studies should facilitate the development of effective new therapies for the treatment of multiple sclerosis that complement established immunosuppressive approaches.

Dopamine Transporter Dynamics of N-Substituted Benztropine Analogs with Atypical Behavioral Effects.

Atypical dopamine transporter (DAT) inhibitors, despite high DAT affinity, do not produce the psychomotor stimulant and abuse profile of standard DAT inhibitors such as cocaine. Proposed contributing features for those differences include off-target actions, slow onsets of action, and ligand bias regarding DAT conformation. Several 3α-(4',4''-difluoro-diphenylmethoxy)tropanes were examined, including those with the following substitutions: N-(indole-3''-ethyl)- (GA1-69), N-(R)-2''-amino-3''-methyl-n-butyl- (GA2-50), N-2''aminoethyl- (GA2-99), and N-(cyclopropylmethyl)- (JHW013). These compounds were previously reported to have rapid onset of behavioral effects and were presently evaluated pharmacologically alone or in combination with cocaine. DAT conformational mode was assessed by substituted-cysteine accessibility and molecular dynamics (MD) simulations. As determined by substituted-cysteine alkylation, all BZT analogs except GA2-99 showed bias for a cytoplasmic-facing DAT conformation, whereas cocaine stabilized the extracellular-facing conformation. MD simulations suggested that several analog-DAT complexes formed stable R85-D476 "outer gate" bonds that close the DAT to extracellular space. GA2-99 diverged from this pattern, yet had effects similar to those of other atypical DAT inhibitors. Apparent DAT association rates of the BZT analogs in vivo were slower than that for cocaine. None of the compounds was self-administered or stimulated locomotion, and each blocked those effects of cocaine. The present findings provide more detail on ligand-induced DAT conformations and indicate that aspects of DAT conformation other than "open" versus "closed" may facilitate predictions of the actions of DAT inhibitors and may promote rational design of potential treatments for psychomotor-stimulant abuse.

Development of Biomarkers for Inhibition of SLC6A19 (B°AT1)-A Potential Target to Treat Metabolic Disorders.

Recent studies have established that dietary protein restriction improves metabolic health and glucose homeostasis. SLC6A19 (B°AT1) is the major neutral amino acid transporter in the intestine and carries out the bulk of amino acid absorption from the diet. Mice lacking SLC6A19 show signs of protein restriction, have improved glucose tolerance, and are protected from diet-induced obesity. Pharmacological blockage of this transporter could be used to induce protein restriction and to treat metabolic diseases such as type 2 diabetes. A few novel inhibitors of SLC6A19 have recently been identified using in vitro compound screening, but it remains unclear whether these compounds block the transporter in vivo. To evaluate the efficacy of SLC6A19 inhibitors biomarkers are required that can reliably detect successful inhibition of the transporter in mice. A gas chromatography mass spectrometry (GC-MS)-based untargeted metabolomics approach was used to discriminate global metabolite profiles in plasma, urine and faecal samples from SLC6A19ko and wt mice. Due to inefficient absorption in the intestine and lack of reabsorption in the kidney, significantly elevated amino acids levels were observed in urine and faecal samples. By contrast, a few neutral amino acids were reduced in the plasma of male SLC6A19ko mice as compared to other biological samples. Metabolites of bacterial protein fermentation such as p-cresol glucuronide and 3-indole-propionic acid were more abundant in SLC6A19ko mice, indicating protein malabsorption of dietary amino acids. Consistently, plasma appearance rates of [14C]-labelled neutral amino acids were delayed in SLC6A19ko mice as compared to wt after intra-gastric administration of a mixture of amino acids. Receiver operating characteristic (ROC) curve analysis was used to validate the potential use of these metabolites as biomarkers. These findings provide putative metabolite biomarkers that can be used to detect protein malabsorption and the inhibition of this transporter in intestine and kidney.

σ Receptor Effects of N-Substituted Benztropine Analogs: Implications for Antagonism of Cocaine Self-Administration.

Several N-substituted benztropine (BZT) analogs are atypical dopamine transport inhibitors as they have affinity for the dopamine transporter (DAT) but have minimal cocaine-like pharmacologic effects and can block numerous effects of cocaine, including its self-administration. Among these compounds, N-methyl (AHN1-055), N-allyl (AHN2-005), and N-butyl (JHW007) analogs of 3α-[bis(4'-fluorophenyl)methoxy]-tropane were more potent in antagonizing self-administration of cocaine and d-methamphetamine than in decreasing food-maintained responding. The antagonism of cocaine self-administration (0.03-1.0 mg/kg per injection) with the above BZT analogs was reproduced in the present study. Further, the stimulant-antagonist effects resembled previously reported effects of pretreatments with combinations of standard DAT inhibitors and σ1-receptor (σ1R) antagonists. Therefore, the present study examined binding of the BZT analogs to σRs, as well as their in vivo σR antagonist effects. Each of the BZT analogs displaced radiolabeled σR ligands with nanomolar affinity. Further, self-administration of the σR agonist DTG (0.1-3.2 mg/kg/injection) was dose dependently blocked by AHN2-005 and JHW007 but potentiated by AHN1-055. In contrast, none of the BZT analogs that were active against DTG self-administration was active against the self-administration of agonists at dopamine D1-like [R(+)-SKF 81297, (±)-SKF 82958 (0.00032-0.01 mg/kg per injection each)], D2-like [R(-)-NPA (0.0001-0.0032 mg/kg per injection), (-)-quinpirole (0.0032-0.1 mg/kg per injection)], or µ-opioid (remifentanil, 0.0001-0.0032 mg/kg per injection) receptors. The present results indicate that behavioral antagonist effects of the N-substituted BZT analogs are specific for abused drugs acting at the DAT and further suggest that σR antagonism contributes to those actions.

Further delineation between typical and atypical dopamine uptake inhibitors: effects on food-maintained behavior and food consumption.

The present studies compared the acute effects of benztropine analogs (4-Cl-BZT, JHW 007, AHN 1-055), which are atypical dopamine uptake inhibitors, with those of the standard dopamine uptake inhibitors GBR 12909 and cocaine, on the reinforcing efficacy of food and food intake in male Sprague-Dawley rats. Repeated drug effects of JHW 007 on food intake were also determined. The number of ratios completed under a progressive-ratio schedule of food delivery was used as an index of reinforcing efficacy. Food intake was determined by measuring powdered laboratory-chow consumption during daily 40 min food-availability time periods. Under the progressive-ratio schedule, cocaine and GBR 12909 dose-dependently increased the number of ratios completed. JHW 007 decreased ratios completed, whereas neither 4-Cl-BZT nor AHN 1-055 increased ratios completed with a magnitude that approximated any of the increases produced by cocaine or GBR 12909. Acute administration of each drug dose-dependently decreased food intake; however, the benztropine analogs were more potent than cocaine and GBR 12909. A reduction in food intake emerged after repeated administration of a low dose of JHW 007. Future studies that compare JHW 007 with standard anorectic drugs (e.g. phentermine) and continue investigation of the repeated drug effects under similar experimental procedures are clearly warranted.

The Transcriptome of Type I Murine Astrocytes under Interferon-Gamma Exposure and Remyelination Stimulus.

Astrocytes are considered to be an important contributor to central nervous system (CNS) disorders, particularly multiple sclerosis. The transcriptome of these cells is greatly affected by cytokines released by lymphocytes, penetrating the blood-brain barrier-in particular, the classical pro-inflammatory cytokine interferon-gamma (IFNγ). We report here the transcriptomal profiling of astrocytes treated using IFNγ and benztropine, a putative remyelinization agent. Our findings indicate that the expression of genes involved in antigen processing and presentation in astrocytes are significantly upregulated upon IFNγ exposure, emphasizing the critical role of this cytokine in the redirection of immune response towards self-antigens. Data reported herein support previous observations that the IFNγ-induced JAK-STAT signaling pathway may be regarded as a valuable target for pharmaceutical interventions.

Inhibition of Ebola and Marburg Virus Entry by G Protein-Coupled Receptor Antagonists.

UNLABELLED: Filoviruses, consisting of Ebola virus (EBOV) and Marburg virus (MARV), are among the most lethal infectious threats to mankind. Infections by these viruses can cause severe hemorrhagic fevers in humans and nonhuman primates with high mortality rates. Since there is currently no vaccine or antiviral therapy approved for humans, there is an urgent need to develop prophylactic and therapeutic options for use during filoviral outbreaks and bioterrorist attacks. One of the ideal targets against filoviral infection and diseases is at the entry step, which is mediated by the filoviral glycoprotein (GP). In this report, we screened a chemical library of small molecules and identified numerous inhibitors, which are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs, including histamine receptors, 5-HT (serotonin) receptors, muscarinic acetylcholine receptor, and adrenergic receptor. These inhibitors can effectively block replication of both infectious EBOV and MARV, indicating a broad antiviral activity of the GPCR antagonists. The time-of-addition experiment and microscopic studies suggest that GPCR antagonists block filoviral entry at a step following the initial attachment but prior to viral/cell membrane fusion. These results strongly suggest that GPCRs play a critical role in filoviral entry and GPCR antagonists can be developed as an effective anti-EBOV/MARV therapy. IMPORTANCE: Infection of Ebola virus and Marburg virus can cause severe illness in humans with a high mortality rate, and currently there is no FDA-approved vaccine or therapeutic treatment available. The 2013-2015 epidemic in West Africa underscores a lack of our understanding in the infection and pathogenesis of these viruses and the urgency of drug discovery and development. In this study, we have identified numerous inhibitors that are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs. These inhibitors can effectively block replication of both infectious EBOV and MARV, indicating a broad antiviral activity of the GPCR antagonists. Our results strongly suggest that GPCRs play a critical role in filoviral entry and GPCR antagonists can be developed as an effective anti-EBOV/MARV therapy.

α-Synuclein-induced myelination deficit defines a novel interventional target for multiple system atrophy.

Multiple system atrophy (MSA) is a rare atypical parkinsonian disorder characterized by a rapidly progressing clinical course and at present without any efficient therapy. Neuropathologically, myelin loss and neurodegeneration are associated with α-synuclein accumulation in oligodendrocytes, but underlying pathomechanisms are poorly understood. Here, we analyzed the impact of oligodendrocytic α-synuclein on the formation of myelin sheaths to define a potential interventional target for MSA. Post-mortem analyses of MSA patients and controls were performed to quantify myelin and oligodendrocyte numbers. As pre-clinical models, we used transgenic MSA mice, a myelinating stem cell-derived oligodendrocyte-neuron co-culture, and primary oligodendrocytes to determine functional consequences of oligodendrocytic α-synuclein overexpression on myelination. We detected myelin loss accompanied by preserved or even increased numbers of oligodendrocytes in post-mortem MSA brains or transgenic mouse forebrains, respectively, indicating an oligodendrocytic dysfunction in myelin formation. Corroborating this observation, overexpression of α-synuclein in primary and stem cell-derived oligodendrocytes severely impaired myelin formation, defining a novel α-synuclein-linked pathomechanism in MSA. We used the pro-myelinating activity of the muscarinic acetylcholine receptor antagonist benztropine to analyze the reversibility of the myelination deficit. Transcriptome profiling of primary pre-myelinating oligodendrocytes demonstrated that benztropine readjusts myelination-related processes such as cholesterol and membrane biogenesis, being compromised by oligodendrocytic α-synuclein. Additionally, benztropine restored the α-synuclein-induced myelination deficit of stem cell-derived oligodendrocytes. Strikingly, benztropine also ameliorated the myelin deficit in transgenic MSA mice, resulting in a prevention of neuronal cell loss. In conclusion, this study defines the α-synuclein-induced myelination deficit as a novel and crucial pathomechanism in MSA. Importantly, the reversible nature of this oligodendrocytic dysfunction opens a novel avenue for an intervention in MSA.

mRNA expression profile of mouse oligodendrocytes in inflammatory conditions.

In this study, we performed transcriptome profiling of oligodendrocyte culture of mice treated with the remyelinating therapeutic agent benztropine in the presence and absence of interferon gamma (IFNγ). The results of this work are important for understanding the expression profile of oligodendrocytes under conditions of systemic inflammation in the central nervous system in multiple sclerosis as well as the mechanisms of cellular response to benztropine in light of its possible use for the treatment of multiple sclerosis.

Preclinical efficacy of N-substituted benztropine analogs as antagonists of methamphetamine self-administration in rats.

Atypical dopamine-uptake inhibitors have low abuse potential and may serve as leads for development of cocaine-abuse treatments. Among them, the benztropine (BZT) derivatives, N-butyl (JHW007), N-allyl (AHN2-005), and N-methyl (AHN1-055) analogs of 3α-[bis(4'-fluorophenyl)methoxy]-tropane dose-dependently decreased cocaine self-administration without effects on food-maintained responding. Our study examined selectivity by assessing their effects on self-administration of other drugs. As with cocaine, each BZT analog (1.0-10.0 mg/kg i.p.) dose-dependently decreased maximal self-administration of d-methamphetamine (0.01-0.32 mg/kg/infusion) but was inactive against heroin (1.0-32.0 µg/kg/infusion) and ketamine (0.032-1.0 mg/kg/infusion) self-administration. Further, standard dopamine indirect-agonists [WIN35,428 ((-)-3ß-(4-fluorophenyl)-tropan-2-ß-carboxylic acid methyl ester tartrate), d-amphetamine (0.1-1.0 mg/kg i.p., each)] dose-dependently left-shifted self-administration dose-effect curves for d-methamphetamine, heroin, and ketamine. Noncompetitive NMDA-glutamate receptor/channel antagonists [(+)-MK-801 (0.01-0.1 mg/kg i.p.), memantine (1.0-10.0 mg/kg i.p.)] also left-shifted dose-effect curves for d-methamphetamine and ketamine (but not heroin) self-administration. The µ-agonists [dl-methadone and morphine (1.0-10.0 mg/kg i.p., each)] dose-dependently decreased maximal self-administration of µ-agonists (heroin, remifentanil) but not d-methamphetamine or ketamine self-administration. The µ-agonist-induced decreases were similar to the effects of BZT analogs on stimulant self-administration and effects of food prefeeding on responding maintained by food reinforcement. Radioligand-binding and behavioral studies suggested that inhibition of dopamine transporters and σ receptors were critical for blocking stimulant self-administration by BZT-analogs. Thus, the present results suggest that the effects of BZT analogs on stimulant self-administration are similar to effects of µ-agonists on µ-agonist self-administration and food prefeeding on food-reinforced responding, which implicates behavioral mechanisms for these effects and further supports development of atypical dopamine uptake inhibitors as medications for stimulant abuse.

Pharmacological characterization of a dopamine transporter ligand that functions as a cocaine antagonist.

An N-butyl analog of benztropine, JHW007 [N-(n-butyl)-3α-[bis(4'-fluorophenyl)methoxy]-tropane], binds to dopamine transporters (DAT) but has reduced cocaine-like behavioral effects and antagonizes various effects of cocaine. The present study further examined mechanisms underlying these effects. Cocaine dose-dependently increased locomotion, whereas JHW007 was minimally effective but increased activity 24 hours after injection. JHW007 (3-10 mg/kg) dose-dependently and fully antagonized the locomotor-stimulant effects of cocaine (5-60 mg/kg), whereas N-methyl and N-allyl analogs and the dopamine (DA) uptake inhibitor GBR12909 [1-(2-[bis(4-fluorophenyl)methoxy]ethyl)-4-(3-phenylpropyl)piperazine dihydrochloride] stimulated activity and failed to antagonize effects of cocaine. JHW007 also blocked the locomotor-stimulant effects of the DAT inhibitor GBR12909 but not stimulation produced by the δ-opioid agonist SNC 80 [4-[(R)-[(2S,5R)-4-allyl-2,5-dimethylpiperazin-1-yl](3-methoxyphenyl)methyl]-N,N-diethylbenzamide], which increases activity through nondopaminergic mechanisms. JHW007 blocked locomotor-stimulant effects of cocaine in both DA D2- and CB1-receptor knockout and wild-type mice, indicating a lack of involvement of these targets. Furthermore, JHW007 blocked effects of cocaine on stereotyped rearing but enhanced stereotyped sniffing, suggesting that interference with locomotion by enhanced stereotypies is not responsible for the cocaine-antagonist effects of JHW007. Time-course data indicate that administration of JHW007 antagonized the locomotor-stimulant effects of cocaine within 10 minutes of injection, whereas occupancy at the DAT, as determined in vivo, did not reach a maximum until 4.5 hours after injection. The σ1-receptor antagonist BD 1008 [N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)ethylamine dihydrobromide] blocked the locomotor-stimulant effects of cocaine. Overall, these findings suggest that JHW007 has cocaine-antagonist effects that are deviate from its DAT occupancy and that some other mechanism, possibly σ-receptor antagonist activity, may contribute to the cocaine-antagonist effect of JHW007 and like drugs.

The effect of anticholinergic drugs on cognition of patients with Parkinson's disease: a cohort study from the Egyptian population.

BACKGROUND: Cognitive dysfunction is a non-motor manifestation of Parkinson's disease (PD). We aimed to determine the frequency and patterns of cognitive dysfunction in treated patients with PD and their predictors. RESEARCH DESIGN AND METHODS: This study included 80 patients (male = 48; female = 32) and 30 healthy individuals. They underwent neuropsychiatric evaluations. Measurements included Beck's depression inventory - II (BDI-II), mini-mental state examination (MMSE) and Montreal cognitive assessment (MoCA). RESULTS: Patients had mean age of 55.56 ± 9.06 yrs, duration of PD of 4.86 ± 2.71 yrs and Hoehn and Yahr Scoring of 2.19 ± 0.89. They were on levodopa/carbidopa therapy and adjuvant therapy with benztropine mesylate, an anticholinergic drug, (n = 51) or amantadine sulfate, a dopaminergic drug, (n = 29). Sixteen (20%) had moderate depressive symptoms. Mild and moderate cognitive impairments were reported in 38.8% and 28.8% (by MMSE) and 46.3% and 31.3% (by MoCA). Patients had lower global cognitive scoring (p = 0.0001) and scorings of different cognitive functions (naming, attention, language, abstraction, memory and orientation) than controls. Patients treated with benztropine had lower cognition than with amantadine. Correlation analyses showed that lower cognition was only associated with chronic PD and its treatment (p = 0.0001). CONCLUSIONS: Cognitive dysfunction is common with PD (77.5%) particularly with anticholinergic drugs. De-prescription of anticholinergics is recommended for patients with PD.

Substituting a long-acting dopamine uptake inhibitor for cocaine prevents relapse to cocaine seeking.

The treatment of cocaine addiction remains a challenge. The dopamine replacement approach in cocaine addiction involves the use of a competing dopaminergic agonist that might suppress withdrawal and drug craving in abstinent individuals. Although it has long been postulated that such an approach may be therapeutically successful, preclinical or clinical evidence showing its effectiveness to prevent relapse is scant. We used in rats a procedure that involved substitution of the N-substituted benztropine analog 3α-[bis(4'-fluorophenyl)methoxy]-tropane (AHN-1055), a long-acting dopamine uptake inhibitor (DUI), for cocaine. Maintenance treatment was self-administered. After extinction, reinstatement of drug seeking was induced by cocaine priming. We measured the contents of brain-derived neurotrophic factor (BDNF), c-Fos and Fas-associated death domain (FADD) proteins in the medial prefrontal cortex (mPFC) following reinstatement. DUI, but not amphetamine, substitution led to extinction of active lever presses, as did saline substitution. DUI substitution significantly reduced cocaine-induced reinstatement of drug-seeking behavior, which was strongly elicited after saline substitution. Rats passively yoked to DUI also showed reduced cocaine-primed reinstatement. Reductions in drug seeking during reinstatement were matched by downward shifts in the contents of BDNF, c-Fos and FADD proteins in the mPFC, which were elevated in relapsing rats. These data indicate that DUI substitution not only leads to extinction of self-administration behavior but also prevents reinstatement of drug seeking induced by cocaine re-exposure. Thus, DUI substitution therapy using compounds with low abuse potential, even if received passively in the context previously paired with drug taking, may provide an effective treatment for stimulant addiction.

Benztropine and suicide attempts and intentional self-harm.

BACKGROUND: This large-scale pharmacoepidemiologic study was conducted to confirm a previous signal for decreased risk of suicide attempt following prescription fills for benztropine. METHODS: We used a within-person exposure-only cohort design to study the dynamic association between benztropine prescription fills over a 12-month period and suicidal events (suicide attempts and intentional self-harm) in 62,493 patients with private health insurance (MarketScan - MS) who filled a new benztropine prescription between 2011 and 2019. A discrete-time survival analysis was used to analyze the data, adjusting for age, sex, diagnoses related to suicidal behavior, Parkinson's disease, medical comorbidities, history of suicide attempts, concomitant CNS medications, and time-varying antipsychotic use. RESULTS: Overall, there were 486 suicidal events (0.8%) following the index end-date of the one-year baseline period. Benztropine use was associated with fewer suicidal events (HR=0.63, 95% CI = 0.50, 0.80). Patients treated with antipsychotics and benztropine had a similar reduction in suicidal events as patients treated with benztropine alone in both within-subject and between-subject analyses. Similar associations were found for patients with bipolar disorder or schizophrenia, and those treated with newer versus older generation antipsychotics. Dose-response and duration response relationships were found, with an overall 6% reduction in suicidal events per 1 mg equivalent dosage per month, that was similar in those treated and those not treated with antipsychotics. INTERPRETATIONS: Benztropine was found to lower suicidal event rates, comparably in those receiving or not receiving antipsychotic medications, regardless of the presence of major psychiatric disorders. This observation warrants testing in a randomized clinical trial. FUNDING: No funding sources were utilized for this manuscript.

Novel C-1 substituted cocaine analogs unlike cocaine or benztropine.

Despite a wealth of information on cocaine-like compounds, there is no information on cocaine analogs with substitutions at C-1. Here, we report on (R)-(-)-cocaine analogs with various C-1 substituents: methyl (2), ethyl (3), n-propyl (4), n-pentyl (5), and phenyl (6). Analog 2 was equipotent to cocaine as an inhibitor of the dopamine transporter (DAT), whereas 3 and 6 were 3- and 10-fold more potent, respectively. None of the analogs, however, stimulated mouse locomotor activity, in contrast to cocaine. Pharmacokinetic assays showed compound 2 occupied mouse brain rapidly, as cocaine itself; moreover, 2 and 6 were behaviorally active in mice in the forced-swim test model of depression and the conditioned place preference test. Analog 2 was a weaker inhibitor of voltage-dependent Na+ channels than cocaine, although 6 was more potent than cocaine, highlighting the need to assay future C-1 analogs for this activity. Receptorome screening indicated few significant binding targets other than the monoamine transporters. Benztropine-like "atypical" DAT inhibitors are known to display reduced cocaine-like locomotor stimulation, presumably by their propensity to interact with an inward-facing transporter conformation. However, 2 and 6, like cocaine, but unlike benztropine, exhibited preferential interaction with an outward-facing conformation upon docking in our DAT homology model. In summary, C-1 cocaine analogs are not cocaine-like in that they are not stimulatory in vivo. However, they are not benztropine-like in binding mechanism and seem to interact with the DAT similarly to cocaine. The present data warrant further consideration of these novel cocaine analogs for antidepressant or cocaine substitution potential.

Blockade of Voltage-Dependent K+ Channels by Benztropine, a Muscarinic Acetylcholine Receptor Inhibitor, in Coronary Arterial Smooth Muscle Cells.

We investigated the effect of the acetylcholine muscarinic receptor inhibitor benztropine on voltage-dependent K+ (Kv) channels in rabbit coronary arterial smooth muscle cells. Benztropine inhibited Kv currents in a concentration-dependent manner, with an apparent IC50 value of 6.11 ± 0.80 µM and Hill coefficient of 0.62 ± 0.03. Benztropine shifted the steady-state activation curves toward a more positive potential, and the steady-state inactivation curves toward a more negative potential, suggesting that benztropine inhibited Kv channels by affecting the channel voltage sensor. Train pulse (1 or 2 Hz)-induced Kv currents were effectively reduced by the benztropine treatment. Furthermore, recovery time constants of Kv current inactivation increased significantly in response to benztropine. These results suggest that benztropine inhibited vascular Kv channels in a use (state)-dependent manner. The inhibitory effect of benztropine was canceled by pretreatment with the Kv 1.5 inhibitor, but there was no obvious change after pretreatment with Kv 2.1 or Kv7 inhibitors. In conclusion, benztropine inhibited the Kv current in a concentration- and use (state)-dependent manner. Inhibition of the Kv channels by benztropine primarily involved the Kv1.5 subtype. Restrictions are required when using benztropine to patients with vascular disease.

Combination of Antimalarial and CNS Drugs with Antineoplastic Agents in MCF-7 Breast and HT-29 Colon Cancer Cells: Biosafety Evaluation and Mechanism of Action.

Drug combination and drug repurposing are two strategies that allow to find novel oncological therapies, in a faster and more economical process. In our previous studies, we developed a novel model of drug combination using antineoplastic and different repurposed drugs. We demonstrated the combinations of doxorubicin (DOX) + artesunate, DOX + chloroquine, paclitaxel (PTX) + fluoxetine, PTX + fluphenazine, and PTX + benztropine induce significant cytotoxicity in Michigan Cancer Foundation-7 (MCF-7) breast cancer cells. Furthermore, it was found that 5-FU + thioridazine and 5-fluorouracil (5-FU) + sertraline can synergistically induce a reduction in the viability of human colorectal adenocarcinoma cell line (HT-29). In this study, we aim to (1) evaluate the biosafety profile of these drug combinations for non-tumoral cells and (2) determine their mechanism of action in cancer cells. To do so, human fetal lung fibroblast cells (MRC-5) fibroblast cells were incubated for 48 h with all drugs, alone and in combination in concentrations of 0.25, 0.5, 1, 2, and 4 times their half-maximal inhibitory concentration (IC50). Cell morphology and viability were evaluated. Next, we designed and constructed a cell microarray to perform immunohistochemistry studies for the evaluation of palmitoyl-protein thioesterase 1 (PPT1), Ki67, cleaved-poly (ADP-ribose) polymerase (cleaved-PARP), multidrug resistance-associated protein 2 (MRP2), P-glycoprotein (P-gp), and nuclear factor-kappa-B (NF-kB) p65 expression. We demonstrate that these combinations are cytotoxic for cancer cells and safe for non-tumoral cells at lower concentrations. Furthermore, it is also demonstrated that PPT1 may have an important role in the mechanism of action of these combinations, as demonstrated by their ability to decrease PPT1 expression. These results support the use of antimalarial and central nervous system (CNS) drugs in combination regimens with chemotherapeutic agents; nevertheless, additional studies are recommended to further explore their complete mechanisms of action.

Therapeutic-like properties of a dopamine uptake inhibitor in animal models of amphetamine addiction.

N-substituted benztropine (BZT) analogs are molecules that display high affinity for the dopamine transporter (DAT), therapeutic-like effects in animal models of cocaine abuse, and psychopharmacological characteristics consistent with those of a substitute medication for cocaine addiction. Since amphetamine (Amph) and cocaine share mechanisms of action at the DAT, we evaluated the effectiveness of a BZT analog in animal models of Amph addiction. We tested in mice and rats the effects of the BZT derivative, 3α-[bis(4-fluorophenyl)methoxy]-tropane (AHN-1055), on Amph-induced conditioned place preference (CPP), locomotor activity, sensitization, self-administration and ΔFosB accumulation in the nucleus accumbens (NAc). The results showed that AHN-1055 did not produce rewarding, stimulant, or sensitized locomotor effects in mice when administered alone but it readily blocked the rewarding, stimulant, and sensitizing effects of repeated Amph exposure. Furthermore, in mice undergoing conditioning in the CPP paradigm, the BZT analog prevented the accumulation of ΔFosB protein induced in the NAc shell region by Amph treatment. Notably, treatment with AHN-1055 dose-dependently reduced Amph self-administration in rats with a steady history of voluntary Amph intake. These results provide a straightforward demonstration that a BZT derivative with binding affinity for DAT exhibits high efficacy in animal models of Amph abuse, suggesting that the novel generation of BZT analogs could have wider therapeutic applications in stimulant-spectrum disorders than those previously recognized.

Human embryonic stem cell-derived cardiomyocyte platform screens inhibitors of SARS-CoV-2 infection.

Patients with cardiovascular comorbidities are more susceptible to severe infection with SARS-CoV-2, known to directly cause pathological damage to cardiovascular tissue. We outline a screening platform using human embryonic stem cell-derived cardiomyocytes, confirmed to express the protein machinery critical for SARS-CoV-2 infection, and a SARS-CoV-2 spike-pseudotyped virus system. The method has allowed us to identify benztropine and DX600 as novel inhibitors of SARS-CoV-2 infection in a clinically relevant stem cell-derived cardiomyocyte line. Discovery of new medicines will be critical for protecting the heart in patients with SARS-CoV-2, and for individuals where vaccination is contraindicated.