Exploring ligand selectivity in monoamine transporters

The serotonin transporter (SERT), the dopamine transporter (DAT) and the norepinephrine transporter (NET) regulate monoaminergic neurotransmission by reuptake of the released monoamines into neurons. Besides being the target for psychostimulants such as amphetamine and cocaine, the transporters for the monoamines are important drug targets in the treatment of various diseases, including depression, anxiety, obesity, drug abuse, obsessive-compulsive disorder, attention deficit hyperactive disorder (ADHD), and sleep disorders. Understanding the binding modes of drugs that affect the monoamine transporters is essential to explain their mechanism of action and to develop new and improved therapeutic compounds. Selective drugs are desired in the treatment of particular diseases. For example, it is beneficial to treat depression by inhibiting SERT but not DAT. Conversely only inhibitors of DAT and NET ameliorate the symptoms of ADHD. Considerable efforts have been made to explore the molecular basis of ligand selectivity in monoamine transporters, which has been so far implicitly assumed to result from differences in dissociation rate constant. However, the study from one of the co-authors of this proposal (Dr. Walter Sandtner, Medical University Vienna) suggests that ligand selectivity for monoamine transporters is in part driven by association rate constant (kon), which implies the existence of a selectivity filter, possibly involving additional ligand interaction sites in the entry pathway leading to the primary binding site. Over the past years, the applicant developed a nano-pharmacological force sensing method which was used to investigate the interaction forces between transporters and ligands at the single molecular level and to extract the energy landscape and the kinetic rate constants of ligand binding. In particular, the two allosterically related ligand binding sites in SERT were identified by using force sensors functionalized with the antidepressant citalopram. With this technique the dissociation rate constants at the central S1 site of SERT were determined which were subsequently confirmed by electrophysiological measurements. Furthermore, the force sensing was able to distinguish the binding events from the two binding sites, thus providing a unique approach for obtaining the kinetic rate constants at the transient vestibular S2 site. This is currently not possible with any alternative technique. In the present proposal we will investigate the molecular mechanisms in SERT and DAT which underlie the kon-based selectivity. We further aim at identifying the structural elements involved in the formation of the proposed selectivity filter. For this purpose we will combine the single molecule force sensing and electrophysiological methods to address the question how these transporters can be selectively targeted by ligands and what is the functional role of the S2 site.

The monoamine transporters (MATs) are targets for psychostimulants and therapeutics used to treat diseases such as depression or attention deficit hyperactive disorder. Understanding the binding modes of drugs that affect the MATs is essential to explain their mechanism of action and to develop new effective ligands. Considerable efforts have been spent to explore the molecular basis of ligand selectivity in MATs, which has generally been assumed to result from differences in the dissociation rate. However, recent study suggests that the ligand selectivity of MATs is in part driven by the association rate, implying the existence of a selectivity filter, possibly involving additional ligand interaction sites in the entry pathway which leads to the primary binding site in MATs. We explored the selectivity filter by using the single molecule force spectroscopy based on atomic force microscopy (AFM) and electrophysiological measurements. Many drug molecules such as desipramine have a secondary amine group instead of a primary amine group, for which there is no published method for functionalization of AFM tip. In this project, we developed the chemistry to conjugate desipramine to AFM tip through the reaction between bromoacetate and the secondary amine of desipramine, which paved a new way for functionalization of AFM tips with drug molecules. From the force measurements we found that the dissociation rate of desipramine was similar for both transporters of dopamine (DAT) and serotonin (SERT). However, the association rate of desipramine for SERT was about six times higher than that for DAT. Therefore, the selectivity of desipramine for SERT over DAT is mainly determined by its association rather than dissociation rate. By using antibody fragments targeting the extracellular loop 2 (EL2) and 4 (EL4), we found that the occupation of EL2 and EL4 reduced the association rate of desipramine for SERT but not for DAT, with negligible effect on the dissociation rate, suggesting that EL2 and EL4 contribute to the selectivity filter which facilitates desipramine entry to the primary binding site of SERT. Utilizing electrophysiological recordings, we extended the exploration of the selectivity filter to methylphenidate which is a selective inhibitor of DAT. The results showed that the EL4 antibody and its fragment impeded the initial substrate-induced transition from the outward to the inward-facing conformation but not the forward cycling mode of SERT. In contrast, occupancy of EL2 with antibody fragment enhanced the affinity of SERT for methylphenidate by accelerating the association rate. This was confirmed by examining binding of certain radiolabeled inhibitors to SERT. Based on all these observations, we conclude that EL2 and EL4 control access of inhibitors to the binding of SERT and DAT, thus acting as a selectivity filter. These insights have repercussions for drug development.