Novel Probes for the Monoamine Transporters

National Institute On Drug Abuse

Investigators

Linked publications, trials & patents

Abstract

The inhibition of dopamine reuptake via the dopamine transporter (DAT) has been characterized as the primary mechanism by which cocaine produces its psychomotor stimulant and reinforcing actions. In order to understand further the molecular mechanisms underlying cocaine use disorder, structure-function studies have been directed toward characterizing the DAT protein at a molecular level. Of note, cocaine binds to an outward facing conformation of the DAT, whereas our atypical DAT inhibitors prefer an inward facing occluded conformation. Previous conformational studies provided evidence, at the molecular level, that the atypical dopamine uptake inhibitors are indeed functioning differently than cocaine at the DAT, and this is related to their distinct behavioral profiles. More recently we have focused attention on modafinil, which binds to the DAT and is currently used clinically for the treatment of sleep disorders. Modafinil has been evaluated as a potential medication to treat methamphetamine and cocaine abuse, with limited success, and is also being used off-label for the treatment of ADHD. Thus far these novel analogs demonstrate a unique SAR profile. Modifications to the modafinil template have resulted in molecules with high affinity (>1000-fold higher than the parent drug) and selective binding to the DAT. In addition, computational studies support experiments in the mutant DATs that suggest that several of our modafinil analogues prefer a more occluded conformation of the DAT, more like our previously and extensively published benztropine analogs than cocaine. In addition, we have discovered cocaine and cocaine-like typical psychostimulants elicit changes in dopamine dynamics distinct from those elicited by the atypical dopamine uptake inhibitors, as measured via voltammetry procedures. Our results suggest a role for CaMKII in modulating the effects of cocaine on evoked DA release without affecting cocaine inhibition of DA reuptake. This effect is related to a specific DAT conformation stabilized by cocaine. Moreover, our atypical DAT inhibitors, which prefer a distinct DAT conformation, blunt cocaines neurochemical and behavioral effects, indicating a unique mechanism underlying their potential as medications for treating psychostimulant use disorder. We have demonstrated that very subtle differences in this structural template can convert an atypical DAT inhibitor (e.g., JJC8-091) into a more typical cocaine-like molecule (e.g., JJC8-088). Metabolism, pharmacokinetic and behavioral analyses identified JJC8-091 as a lead atypical DAT inhibitor for further development. Nevertheless, in a recent drug-food choice model in nonhuman primates, JJC8-088 was able to reduce both food and cocaine intake, whereas JJC8-091 had no effect on these behaviors. JJC8-091 demonstrated lower binding affinity in monkey brain than in rat brain, which may have resulted in limited DAT binding in vivo. Current drug design efforts have been focused on improving binding affinities of the atypical DAT inhibitors and also investigating whether or not mild stimulant-like activity is necessary for effectiveness in these models. More recently several new analogues, including RDS03-94 and RDS4-010 have been evaluated in rodent models of cocaine misuse. These subtle structural changes at the molecular level can profoundly change the behavioral profile of these molecules and these insights at the atomistic level, have led to novel drug design. In addition, we have focused our drug design toward improving both metabolic stability and reducing hERG channel activity, a predictor of cardiotoxicity. This work is leading to new small molecules that have both the desired pharmacological profiles as well as drug-like properties that will improve their chances of translation to the clinic. In addition to medication potential for PSUD, atypical DAT inhibitors may also serve as pharmacochaperones that may mitigate the severely disabling motor disorders associated with human genetic mutations in DAT. Noribogaine has recently been described as a pharmacochaperone, capable of rescuing mutant DATs that are unable to fold properly in the endoplasmic reticulum, so never make it to the membrane, leaving the patient with poor DAT function that results in movement and neuropsychiatric disorders. Hence, we have embarked on a screening and synthesis project to identify lead molecules that may rescue these mutant DATs and provide the opportunity to improve DAT function and ameliorate symptoms associated with these disorders. We have identified a series of deconstructed ibogaine analogues, synthesized in our lab, that exhibit DAT and SERT pharmacochaperoning activities that surpass those of noribogaine, with our current lead being DG4-69. Further molecular pharmacology and computational modeling studies are underway to advance our understanding of the mechanistic underpinnings of these actions, as well as in vivo studies. In addition to developing agents for in vivo studies, we have also synthesized a number of important bioconjugate molecular tools directed toward the monoamine transporters. Our fluorescent tropane-based ligand, JHC1-064, has been used in many labs to characterize the trafficking and cellular distribution of SERT, NET and DAT in living neuronal cells. More recently, we have designed novel fluorescent ligands, using customized fluorophores suitable for live super resolution imaging. Modification of the linker between the tropane pharmacophore and the fluorophore as well as replacing the rhodamine of JHC1-064 with super bright JaneliaFluor (JF) fluorophores has resulted in the novel fluorescent ligands DG3-80 and DG4-91 that have been used to visualize DAT using super resolution microscopy. In addition, MFZ9-18, an Oregon Green-labeled tropane-based fluorophore, was recently used to visualize dopaminergic axons in rhesus macaque brain tissue slices enabling patch clamp electrophysiology experiments to be performed. We have now synthesized a new series of fluorescent ligands based on our modafinil analogues, JJC8-091 and JJC8-088, using modified linkers and fluorophores to add to our toolbox of fluorescent tools for DAT. Moreover, novel fluorescent ligands based on the NET inhibitors talopram, nisoxetine and methylphenidate were also synthesized, with one nisoxetine analogue showing excellent NET selectivity. The methylphenidate analog was discovered to be suitable for FRET-based assays for Synapsin-III binding. Moreover, our nisoxetine-based fluorescent probe,AC1-146 was used in a study to provide evidence that PIP2 directly regulates NET organization and function. A dysregulation of norepinephrine and PIP2 signaling have both been implicated in neuropsychiatric and cardiovascular diseases.