Research in drug development meets many challenges including lengthy, complex and costly procedures to identify novel pharmacotherapies. In our lab, we developed a method for fast screening of small molecules that interact with monoamine transports – dopamine and serotonin (DAT, SERT). These membrane proteins play important roles in brain neurotransmission responsible for cognition, motion and pleasure. Dysfunction in dopaminergic and serotonergic systems result in neurological disorders such as depression, Attention Deficit Hyperactivity Disorder (ADHD), schizophrenia and addiction. DAT and SERT are responsible for uptake of dopamine (DA) or serotonin (5HT) into the synapse and they limit neurotransmitter signaling. Drugs that mimic or antagonize actions of endogenous neurotransmitters (DA and 5HT) increase the concentrations of DA and/or 5HT either by blocking the transporter (blockers) or by competing uptake with neurotransmitter (substrate). The uptake of substrates is associated to an inward current that depolarizes the cell membrane. Voltage-gated calcium channels (CaV) can respond to small changes in membrane potential. In our method, we combined permanent cell line expressing the human dopamine transporter (hDAT) or the human serotonin transporter (hSERT) (FlpIn TREx expression system) with transient transfection of CaV. This system works as a tightly electrically coupled system. Cells challenged with substrate of the transports produce detectable Ca2+ signal while monoamine transporter blockers can inhibit these Ca2+ signals. The novelty of this method relies on the ability to discriminate between substrate and blockers of monoamine transporters. Preliminary experiments measuring our optimized cell system in a Flex Station 3 plate reader suggest that the co-expression of a voltage-gated Ca2+ channel, a monoamine transporter and a genetically encoded Ca2+ sensor constitute a rapid screening biosensor to identify active drugs at monoamine transporters. Our novel methodology can rapidly assess drug-effect profile on monoamine transporters and benefit development of new psychotherapeutics for treatment of mental illnesses. It can also be used to characterize mechanism of action of emerging drug of abuse, as well as to discover small molecules with novel drug-effect profile useful in basic neuroscience research.

As anfetaminas, grupo de moléculas derivadas da anfetamina, são fármacos estimulantes do sistema nervoso central, e possuem, entre outras, atividade antidepressiva sendo sua ação baseada na inativação da enzima monoamina oxidase (MAO) a qual catalisa a desaminação oxidativa de neurotransmissores, como por exemplo, a serotonina e a noradrenalina. Esta enzima pode ser encontrada em duas diferentes isoformas, a MAO A e MAO B. Este trabalho teve como objetivo o estudo de uma série de derivados anfetamínicos os quais apresentam diferente seletividade e diferentes valores de IC50, variando desde moléculas muito potentes, pouco potentes, até inativas, através da aplicação de técnicas de química-quântica no cálculo de descritores moleculares, bem como a aplicação da mecânica clássica na descrição das interações ligante-receptor a partir de estudos de docking e simulações de dinâmica molecular. Inicialmente, foram aplicados os métodos de química-quântica AM1, HF, DFT (com os funcionais B3LYP e BP86) e MP2 para a determinação do nível de teoria mais apropriado para a otimização da geometria desta série de compostos a partir da comparação entre os resultados teóricos e de raios-X obtidos para a molécula MDMA. O método HF/6-31G(d,p) mostrou os melhores resultados. Desta forma, este método foi utilizado na obtenção das geometrias de mínimo das demais moléculas em estudo. Posteriormente, estes métodos foram utilizados no cálculo das propriedades estruturais, eletrônicas e físico-químicas, distribuição de cargas derivadas do potencial eletrostático, momento dipolar, energia total, energia dos orbitais de fronteira, GAP de energia entre o orbital ocupado de mais alta energia (HOMO) e o orbital desocupado de mais baixa energia (LUMO), dureza, potencial químico, eletronegatividade, eletronegatividade absoluta e eletrofilicidade. Assim, foram verificadas possíveis relações entre os descritores calculados e a atividade biológica determinada por Scorza et al, Hurtado-Guzmán et al and Sterling et al.esta para esta série de compostos. A análise destes resultados foi feita através da aplicação da técnica quimiométrica de análise de componentes principais para, desta forma, verificar-se o agrupamento dos compostos segundo a presença ou ausência de atividade biológica na série estudada. Entretanto, não foi possível se identificar um padrão de agrupamento para as moléculas ativas e inativas, sugerindo assim, que estes descritores não são os mais adequados para a descrição da atividade antidepressiva destes compostos. Em uma segunda etapa, foram feitos estudos de docking para seis diferentes estruturas da MAO B (1OJA, 1OJ9, 2BK3, 2V5Z, 2V60 e 2V61) e cinco da MAO A (2BXR, 2BXS, 2Z5X e 2Z5Y) todas disponíveis no banco de dados de proteína, PDB, no qual se avaliou o modo de interação do ligante no sitio ativo da proteína. Os resultados de docking para a MAO B mostraram que o tamanho do inibidor é importante para uma correta interação no sítio ativo da enzima, tendo-se em vista que o tamanho da cavidade catalítica é dependente da conformação do aminoácido isoleucina 199 e, que a conformação deste aminoácido está relacionada com o tamanho do ligante. Desta forma, a escolha correta da estrutura da proteína torna-se importante para uma correta descrição do sistema. Os resultados sugerem ainda que o átomo do ligante, no qual ocorre a reação com o receptor, deve estar a uma distância média de 3,5 Å do sitio reativo. Os resultados de MD mostraram que este aminoácido é flexível na ausência de um inibidor, onde sua conformação varia entre as chamadas formas aberta e fechada ao longo do tempo, porém, quando o estudo é feito na presença de um ligante, sua conformação perrnanece, de modo geral, constante. / The amphetamine family of drugs is central nervous system stimulant drugs. Amphetamine inhibits the monoamine oxidase enzyme (MAO, isoforms A and B) which catalyzes the oxidative deamination of the neurotransmitter, e. g., serotonin and noradrenalin. In the present study, the aim was to understand the main features of a series of amphetamines derivatives, which have different substrate selectivity and a good range of activity varying from very potent to low potent compounds, even inactive molecules by the application of quantum chemistry techniques to calculate the molecular descriptors, as well as the application of a classical mechanics to describe the ligand-receptor interactions from docking studies and molecular dynamics (MD) simulation. First of all, was applied the AM1, HF, DFT (B3LYP and BP86 functionals) and MP2 quantum chemical methods to analyze which theoretical level is more appropriated for the molecular geometry optimization of this series of compounds from a comparison between the theoretical results for MDMA molecule and it\'s X-ray data. The HF/6-3lG** calculations produced results in close agreement with X-ray crystallography. Thus, was employed the same method for the molecular optimization of the other compounds in the series under investigation. Furthermore, the same method was applied to calculate quantum-chemical parameters (atomic charges, total energy, highest occupied molecular orbital -HOMO- lowest unoccupied molecular orbital -LUMO- dipole moments, hardness, electronegativity, chemical potential, absolute electronegativity and electrophilicity). It was examined possible correlations between the theoretical parameters as calculated by us with the biological activity results as reported by Scorza et al, Hurtado-Guzman et al and Sterling et al. The chemometric technique of Principal Component Analysis for the quantum chemical parameters of these compounds was employed in order to identify some pattern of grouping between the active and inactive molecules. However, it was not possible to identify a pattern between active and inactive compounds suggesting that these above-mentioned parameters are not the best descriptors to evaluate the antidepressant activity for this group of molecules. Later, a docking study was performed for six different PDB structure of MAO B (1OJA, 1OJ9, 2BK3, 2V5Z, 2V60 e 2V61) and five different structure of MAO A (2BXR, 2BXS, 2Z5X e 2Z5Y). It was possible to analyze the ligand-receptor interaction and, according to the bind site size, the activity molecules showed a 3.5A distance between the reactive atoms of the inhibitor and of the protein. Because the conformation of the isoleucine 199 (Ile 199) amino acid can change, the chosen of the correct PDB structure is important for a write description of that interaction. The MD results for the 1OJA structure showed that the Ile199 is flexible in the absence of an inhibitor, where its conformation varies between so-called closed and open forms over the simulation time. However, when the study was done in the presence of a ligand, its conformation remains by generally constant.

Thirty-one rats were trained on a differential reinforcement of low rate schedule. After responding had stabilized, animals were injected with methylphenidate, twice weekly, presession. Methylphenidate produced dose-dependent increases in response rates and decreases in reinforcements. Repetition of these doses produced a reduced drug effect, and a third administration of the 10 mg/kg dose further reduced the drug effect. Subsequently, the effects of daily and intermittent administration were determined for this dose. Daily methylphenidate, pre-session, produced tolerance to the behavioral effects of methylphenidate and cross-tolerance to the amphetamines. Twice-weekly methylphenidate, pre-session, produced partial tolerance to methylphenidate and partial cross-tolerance to the amphetamines. Thus, periodic exposure to the behaviorally disruptive effects of a drug of the amphetamine class reduces the effects of subsequent exposure.

The perinate is particularly risk-prone to chemical species which have the potential of inducing neuronal apoptosis or necrosis and thereby adversely altering development of the brain, to produce life-long functional and behavioral deficits. This paper is an overview for many substances of abuse, but the purview is much more broadened by the realization that even elevated levels of estrogens and corticosteroids in the pregnant mother can act as neuroteratogens, by passing via the placenta and altering neural development or inducing apoptosis in the perinate. Finally, therapeutic risks of anesthetics are highlighted, as these too induce neuronal apoptosis in the neonate by either blocking N-methyl-D-aspartate receptors or by acting as gamma-aminobutyric acid agonists. By understanding the mechanisms involved it may ultimately be possible to interrupt the mechanistic scheme and thereby prevent neuroteratological processes.

Animal models have previously been used to study tolerance and sensitization using two different procedures that are difficult to compare. Tolerance has been studied by administering a drug to a subject that is engaged in an operant behavior, and sensitization by administering a drug to a subject that is not engaged in an operant behavior. Previous research has shown that sensitization can occur when d-amphetamine is administered to rats emitting an operant behavior for a food presentation. The first goal of the experiment was to show operant sensitization using dose response curves. The second goal of the present experiment was to determine if operant sensitization is context specific. These goals were addressed by administering d-amphetamine to rats engaged in an operant behavior in two stimulus contexts and creating dose-response curves. Sensitization occurred but was not found to be context-specific, with the dose-response curves not being significantly different between the two contexts. It is not clear whether this result was due to the drug administration procedure or the counterbalancing assignments used. Further research is needed to determine whether operant sensitization is context specific.

Yes / Background: Asenapine is a new pharmacological agent for the acute treatment of schizophrenia and bipolar disorder. It has relatively higher affinity for serotonergic and α2-adrenergic than dopaminergic D2 receptors. We evaluated the effects of asenapine, risperidone, and olanzapine on acute and subchronic psychotomimetic-induced disruption of cued reversal learning in rats. Methods: After operant training, rats were treated acutely with D-amphetamine (0.75 mg/kg intraperitoneally [i.p.]) or phencyclidine (PCP; 1.5 mg/kg i.p.) or sub-chronically with PCP (2 mg/kg i.p. for 7 days). We assessed the effects of acute coadministration of asenapine, risperidone, or olanzapine on acute D-amphetamine– and PCP-induced deficits and the effects of long-term coadministration of these agents (for 28 additional days) on the deficits induced by subchronic PCP. Results: Deficits in reversal learning induced by acute D-amphetamine were attenuated by risperidone (0.2 mg/kg i.p.). Acute PCP-induced impairment of reversal learning was attenuated by acute asenapine (0.025 mg/kg subcutaneously [s.c.]), risperidone (0.2 mg/kg i.p.), and olanzapine (1.0 mg/kg i.p.). Subchronic PCP administration induced an enduring deficit that was attenuated by acute asenapine (0.075 mg/kg s.c.) and by olanzapine (1.5 mg/kg i.p.). Asenapine (0.075 mg/kg s.c.), risperidone (0.2 mg/kg i.p.), and olanzapine (1.0 mg/kg i.p.) all showed sustained efficacy with chronic (29 d) treatment to improve subchronic PCP-induced impairments. Conclusion: These data suggest that asenapine may have beneficial effects in the treatment of cognitive symptoms in schizophrenia. However, this remains to be validated by further clinical evaluation. / This research was supported by Schering-Plough Corporation, now Merck & Co., Inc. and Pfizer Inc.

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Objective: To investigate whether there is an association between use of cocaine, amphetamines, or psychedelics and psychotic symptoms. Method: Cumulated data from a prospective, longitudinal community study of 2588 adolescents and young adults in Munich, Germany were used. Substance use was assessed at baseline, 4-year and 10-year follow-up using the Munich Composite International Diagnostic Interview; psychotic symptoms were assessed at 4-year and 10-year follow-up. Multinomial logistic regression analyses, adjusted for sociodemographic factors, common mental disorders, other substance use, and childhood adversity (adjusted odds ratios, AOR), revealed associations between cocaine, amphetamine or psychedelic use and psychotic symptoms. Results: Lifetime experience of psychotic symptoms was associated with lifetime use of cocaine (AOR 1.94; 95%CI 1.10-3.45), amphetamines (AOR 1.69; 95%CI 0.98-2.93), psychedelics (AOR 2.37; 95%CI 1.20-4.66) and all three substances (AOR 1.95; 95%CI 1.19-3.18). Conclusion: Associations between psychotic symptoms and use of cocaine, amphetamines, and/or psychedelics in adolescents and young adults call for further studies to elucidate risk factors and developmental pathways.