Methylphenidate, but not citalopram, decreases impulsive choice in rats performing a temporal discounting task.

Introduction: Drugs targeting monoamine systems remain the most common treatment for disorders with impulse control impairments. There is a body of literature suggesting that drugs affecting serotonin reuptake and dopamine reuptake can modulate distinct aspects of impulsivity - though such tests are often performed using distinct behavioral tasks prohibiting easy comparisons. Methods: Here, we directly compare pharmacologic agents that affect dopamine (methylphenidate) vs serotonin (citalopram) manipulations on choice impulsivity in a temporal discounting task where rats could choose between a small, immediate reward or a large reward delayed at either 2 or 10s. In control conditions, rats preferred the large reward at a small (2s) delay and discounted the large reward at a long (10s) delay. Results: Methylphenidate, a dopamine transport inhibitor that blocks reuptake of dopamine, dose-dependently increased large reward preference in the long delay (10s) block. Citalopram, a selective serotonin reuptake inhibitor, had no effect on temporal discounting behavior. Impulsive behavior on the temporal discounting task was at least partially mediated by the nucleus accumbens shell. Bilateral lesions to the nucleus accumbens shell reduced choice impulsivity during the long delay (10s) block. Following lesions, methylphenidate did not impact impulsivity. Discussion: Our results suggest that striatal dopaminergic systems modulate choice impulsivity via actions within the nucleus accumbens shell, whereas serotonin systems may regulate different aspects of behavioral inhibition/impulsivity.

Identifying amino acid residues that contribute to the cellular-DNA binding site on retroviral integrase.

Although retroviral integrase specifically trims the ends of viral DNA and inserts these ends into any sequence in cellular DNA, little information is available to explain how integrase distinguishes between its two DNA substrates. We recently described novel integrase mutants that were improved for specific nicking of viral DNA but impaired at joining these ends into nonviral DNA. An acidic or bulky substitution at one particular residue was critical for this activity profile, and the prototypic protein--Rous sarcoma virus integrase with an S124D substitution--was defective at nonspecifically binding DNA. We have now characterized 19 (including 16 new) mutants that contain one or more aspartic acid substitutions at residues that extend over the surface of the protein and might participate with residue 124 in binding cellular DNA. In particular, every mutant with an aspartate substitution at residue 98 or 128, similar to the original S124D protein, showed improved specific nicking of viral DNA but disturbed nonspecific nicking of nonviral DNA. These data describe a probable cellular-DNA binding platform that involves at least 5 amino acids, in the following order of importance: 124>128>(98, 125)>123. These experimental data are vital for new models of integrase and will contribute to identifying targets for the next generation of integrase inhibitors.

Determination of log D via automated microfluidic liquid-liquid extraction.

A method for log D (pH 7.4) measurement was developed using microfluidic liquid-liquid extraction. Values were determined for 26 compounds and compared to results obtained via shake-flask methods. Excellent correlation between the values obtained via both methods was achieved (R(2) = 0.994). The developed methodology is amenable to automation, enabling high-throughput determination of large compound collections.

Retroviral integrases that are improved for processing but impaired for joining.

Retroviral integrase specifically trims (or processes) the ends of retroviral DNA, then inserts (or joins) these ends into cellular DNA nonspecifically. We previously showed that Rous sarcoma virus integrase with a serine-to-aspartate substitution at amino acid 124 was markedly improved for processing but dramatically impaired for joining, making it the first mutant to separate the activities of integrase in this way. We now show that placing glutamic acid at this residue has the same effect, whereas asparagine or glutamine, which resemble aspartate and glutamate but without the negatively charged acid group, improved processing and impaired joining to a lesser extent. Placing aspartic acid at either of the adjacent residues 123 or 125 also had an intermediate effect. Thus, the charge, structure, and position of the substitution all contribute to the properties of the S124D protein. Infectivity of virions containing these mutations paralleled the in vitro findings, with substitutions having the greatest effect on joining completely blocking replication. Additional studies indicated the replication-defective viruses were blocked at integration and that the S124D protein is impaired at binding nonviral DNA. These functional, biochemical, and genetic data implicate this particular integrase residue as a key part of the binding site for cellular DNA.