The dopamine hypothesis of psychosis developed in the mid-20^th century according to a correlation observed by early investigators showing first generation, or typical, antipsychotics chlorpromazine and haloperidol to antagonize D2 dopamine receptors (D2R). The role of dopamine in psychoses was further characterized by studies with reserpine, which was shown to deplete tissue concentrations of dopamine by a different mechanism later discovered to be the inhibition of vesicular monoamine transporter (VMAT) uptake (Goodman 2011). Data was compiled from sampling tissue from the striatum (caudo-putamen) of the basal ganglia for monoamine concentration, monoaminergic metabolites such as normetanephrine, 3-methoxytyramine, and homovanillic acid and later isolated for radioligand assays (Carlsson 1963, Haracz 1982). The striatum has a high content of GABAergic medium spiny neurons (MSNs) that express either excitatory D1 dopamine receptors or inhibitory D2 dopamine receptors. The basal ganglia exhibit two well-studied cortico-striatal-thalamo-cortical routes that are influenced by dopaminergic activity of the Substantia Nigra Pars Compacta (SNPc) and Ventral Tegmentum Area (VTA) (Figure 1). As GABAergic MSNs in the striatum receive excitatory glutamate neurotransmission from the cortex, MSNs expressing D1 receptors project directly to the Globus Pallidus Interna (GPi) and Substantia Nigra Pars Reticula (SNPr) to influence their GABAergic projection to the thalamus. Meanwhile, GABAergic MSNs expressing D2 receptors project to the Globus Pallidus Externa (GPe), whose GABAergic projections to the Glutamatergic neurons of the Subthalamic Nucleus (STN) regulate the STN’s excitatory input to the GPi and SNPr. The former pathway is known as the Direct Pathway and the latter is known as the Indirect Pathway. Studies have also shown the presence of cholinergic Tonically Active Neurons (TANs) in the striatum that express D2 receptors and project to inhibitory muscarinic M4 receptors on MSNs of the direct pathway and excitatory muscarinic M1 receptors on MSNs of the indirect pathway to further compensate for the action of dopamine as the indirect pathway is favored in conditions of low dopamine, whereas the direct pathway is favored after dopamine release (Bullock 2016).

Figure 1 - Schematic of Basal Ganglia- Adapted from Goodman & Gillman's (2011) and D. Bullock (2016)

It is widely shared hypothesis that persistent D2R antagonism by antipsychotics causes the upregulation and increased sensitivity of D2Rs in the central nervous system (CNS), primarily in the basal ganglia, which encourages the development of EPS (Moncrieff 2009). Moreover, it is suggested that the use of second generation, or atypical, antipsychotics, will exhibit a decrease in EPS risk as they generally have a lower affinity for D2Rs than typical antipsychotics (Moncrieff 2009). However, typical and atypical antipsychotics both demonstrate affinities for various other targets including dopamine (D1 and D4), serotonin (5HT1A, 5HT2A , 5HT2C) histamine (H1), muscarinic and adrenergic receptors that may contribute to therapy as well as side-effects. Recent work by Mahmoudi et al. followed recent findings of D3 dopamine receptor affinity for antipsychotics (haloperidol and clozapine) and performed radioligand-binding assays for D1, D2, and D3 receptors in capuchin monkey basal ganglia that showed upregulation of D3R activity after 6 months treatment with haloperidol in comparison to control; the marginal change in sensitivity of D2 receptors provides evidence of species-related bias of the D2 receptor super-sensitivity hypothesis currently based on rodent models (Mahmoudi et al. 2014). Nevertheless, D2R antagonism may also inhibit the regulative properties of dopaminergic auto-receptors at the presynaptic terminal that act to repolarize the neuron via activation of G-protein inwardly rectifying potassium channels (GIRK) and inhibition of voltage-gated calcium channels (VGCC), as well as providing inhibition to the adenyl cyclase cascade involved in tyrosine hydroxylase activity, an important enzyme in dopamine synthesis (Ford 2014) (Figure 2). Therefore, excess extracellular dopamine concentration remains consequential to antipsychotic use and the therapeutic role of reducing dopamine release via VMAT inhibition presents a viable target.

Figure 2- Dopaminergic Synapse of Basal Ganglia - Adapted from Ford (2014) and Goodman (2011)

Sequence homology studies in mammals indicate Vesicular Monoamine Transporters (VMATs) belong to a subfamily of the major facilitator family proteins and are 12 transmembrane domain proteins present on synaptic vesicles that provide sorting and storage functions of cytosolic monoamines as well as providing the mechanism for exocytotic neurotransmission (Bernstein 2014). Distinct genetic instruction of Solute Carrier 18 subfamily proteins is specific to two isoforms of VMAT, known as VMAT1 and VMAT2 and expression and PET imaging studies confirm VMAT1 is localized to the periphery while VMAT2, which is also expressed in the periphery, is the only isoform expressed in the CNS, with dense populations in monoaminergic neurons of the basal ganglia (Bernstein 2014, Wimalasena 2011). Common to vesicular transporters are V-type H+-ATPase pumps that generate an increased proton gradient within the vesicular matrix that is essential for ligand transport into the vesicle, however this mechanism presents a unique challenge to structure activity relationship studies as transport is dependent on the efflux of two protons and VMAT undergoes a different conformational change after each; the first proton exchange increases the receptors affinity for the ligand while the second proton exchange generates the motive force for ligand internalization with a decrease in ligand binding affinity (Wimalasena 2011). Mutagenesis studies have employed inhibitors Reserpine and Tetrabenazine to distinguish the role of specific residues significant to substrate and inhibitor affinity, especially during the different conformations by varying the available ATP and proton concentration. Combining the understanding that phenylglyoxal and diethyl pryocarbonate interact with histidine residues and showed to inhibit VMAT uptake of serotonin in chromaffin cells, Shirvan et al. showed replacement of conserved Histidine 419 to Arginine or Cystine inhibits serotonin (substrate) uptake and reserpine (inhibitor) binding, suggesting the residues role in proton translocation or forming the energized substrate binding pocket after efflux of the first proton (Shirvan 1994). Conversely, replacement of conserved Aspartate residues 431 and 403 to serine, glutamine or cysteine impeded serotonin uptake while showing little influence on reserpine binding, suggesting the residues role in the second translocation of a proton during uptake (Steiner-Mordoch 1996).

Reserpine has shown to be an irreversible non-selective competitive inhibitor of VMATs 1 and 2, while Tetrabenazine has demonstrated a reversible affinity specifically for VMAT2. Wimalasena et al. further investigated evidence that N-methyl-4-phenyl-tetrahydropyridinium (MPTP) toxicity, specifically via it’s metabolite N-methyl-4-pyridinium (MPP⁺), may facilitate cytosolic monoamine metabolism by blocking VMAT uptake and developed a library of synthesized analogs of MPTP and MPP⁺ with various substituents including the previously defined inhibitor 3-amino-2-phenyl-propene (APP) (Figure 3). Inhibition assays to dopamine on resealed chromaffin granular ghost VMAT revealed that, where a meta-hydroxy substituent on MPP+ (3’OHMPP⁺I^-) demonstrated the greatest inhibition properties of MPP+ derivates (K_i=2.4±0.1 µM), an MPP⁺-APP derivative with meta-hydroxy and large hydrophobic halogen in the para position of the 2-phenylpropene demonstrated the highest inhibition (K_i=0.4±0.1 µM); the investigators further obtained a crystallographic structure of the MPP⁺-APP derivative and used computer modeling to overlay the resulting L-shape conformation onto Tetrabenazine and Reserpine to identify steric agreement and functional groups possibly contributing to affinity such as the 3’ or 4’ OMe substitutions on the MPP+ phenyl, a regionally similar nitrogen that may support ideal positioning of the nitrogen and carbonyl group on Tetrabenazine , and the similar hydrophobic tail may support an important role of the trimethoxy-phenyl tail of Reserpine (Wimalasena 2008).

Figure 3- Structures of SAR Inhibitors from Wimalasena (2008)