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Inhibition of the ternary protein complex of the synaptic scaffolding protein postsynaptic density protein-95 (PSD-95), neuronal nitric oxide synthase (nNOS), and the N-methyl-D-aspartate (NMDA) receptor is a potential strategy for treating ischemic brain damage, but high-affinity inhibitors are lacking. Here we report the design and synthesis of a novel dimeric inhibitor, Tat-NPEG4ðIETDVÞ 2 (Tat-N-dimer), which binds the tandem PDZ1-2 domain of PSD-95 with an unprecedented high affinity of 4.6 nM, and displays extensive protease-resistance as evaluated in vitro by stability-measurements in human blood plasma. X-ray crystallography, NMR, and small-angle X-ray scattering (SAXS) deduced a true bivalent interaction between dimeric inhibitor and PDZ1-2, and also provided a dynamic model of the conformational changes of PDZ1-2 induced by the dimeric inhibitor. A single intravenous injection of Tat-N-dimer (3 nmol∕g) to mice subjected to focal cerebral ischemia reduces infarct volume with 40% and restores motor functions. Thus, Tat-Ndimer is a highly efficacious neuroprotective agent with therapeutic potential in stroke.drug discovery | ischemic stroke | protein-protein interactions P rotein-protein interactions mediated by postsynaptic density protein-95 (PSD-95)/Discs-large/ZO-1 (PDZ) domains are important for intracellular signaling events, and several PDZ domains are potential drug targets for neuronal diseases and cancer (1, 2). The postsynaptic scaffolding protein PSD-95 simultaneously binds the N-methyl-D-aspartate (NMDA)-type of ionotropic glutamate receptors and the enzyme neuronal nitric oxide synthase (nNOS) through its PDZ1 and PDZ2 domains (3). Activation of the NMDA receptor causes influx of Ca 2þ , which activates nNOS thereby leading to nitric oxide generation (4), a key facilitator of glutamate-mediated excitotoxicity (5, 6). Ligands that bind to the first two PDZ domains of PSD-95 inhibit the formation of the ternary nNOS/PSD-95/NMDA receptor complex and uncouple the harmful production of nitric oxide from NMDA receptor activity (Fig. 1A). As PSD-95 inhibition does not affect ion-flux (7) or prosurvival signaling pathways (8) mediated by the NMDA receptor, it is believed that compounds targeting PDZ1 and PDZ2 of PSD-95 can provide an efficient and safe treatment of ischemic brain damage (9), where excitotoxicity is known to dominate in the acute poststroke period, as well as other NMDA receptor-related disorders such as chronic pain and Alzheimer's disease (10-14).The shallow and elongated binding pocket of PDZ domains generally favor binding of peptides or peptide analogues and so far no drug-like small-molecule inhibitors of PDZ domains with affinities below 5 μM have been identified (15). Accordingly, the most advanced PSD-95 inhibitor is a 20-mer peptide, Tat-NR2B9c (7, 8, 16), composed of nine amino acids corresponding to the C-terminal of the GluN2B subunit of the NMDA receptor, fused to the HIV-1 Tat peptide (17). This peptide has shown promising effects against ischemic brain damage in rats (...

The serotonin transporter (SERT)2 is an integral membrane protein that facilitates transport of serotonin (5-hydroxytryptamine, 5HT) across cellular membranes (1). In addition to peripheral endocrine functions, 5HT is a neurotransmitter in the brain; it is involved in control of several important physiological functions such as mood, appetite, and sexual behavior. Expressed mainly in the membrane of serotonergic neurons, SERT utilizes energetically favorable cotransport of Na ϩ to remove released 5HT from the extracellular space. Human SERT (hSERT) belongs to the solute carrier 6 (SLC6) transporter family along with highly homologous transporters for the neurotransmitters ␥-aminobutyric acid, glycine, dopamine, and norepinephrine (2-4). These transporters are important drug targets for treatment of a wide range of neurological diseases. In particular, hSERT is the molecular target for widely used drugs for treatment of depression and anxiety. Also, psychostimulants such as amphetamine and 3,4-methylenedioxy-N-methylamphetamine ("ecstasy") have hSERT as the molecular target (5-7). The selective serotonin re-uptake inhibitors (SSRIs) are a class of antidepressant and anti-anxiety drugs that function as highly selective competitive inhibitors of hSERT (8). Although SSRIs are highly important for treatment of affective disorders (6), the molecular basis for their function, including location and structure of drug binding pockets, is largely unknown and a matter of debate (9, 10). Such information is important for understanding essential aspects of drug action, ranging from selectivity profile to therapeutic efficacy. Moreover, such information is indispensable for the development of new and improved drugs targeting hSERT. The primary impediment for elucidation of the structural mechanisms of hSERT inhibition is the lack of a three-dimensional structure of the protein. Still, several residues in SERT have been identified mainly by mutagenesis studies that modulate antidepressants potency (11)(12)(13)(14)(15)(16)(17). The use of comparative molecular modeling to create structural models of ligand-hSERT interactions has previously been hampered by the low phylogenetic and functional similarity between hSERT and available template proteins (18 -21). However, high resolution crystal structures of a bacterial homolog to mammalian SLC6 transporters, LeuT (22,23), have proven excellent templates for constructing experimentally validated models of substrate and drug binding pockets in human SLC6 transporters, including the human transporters for dopamine and ␥-aminobutyric acid (24 -32).In this study, we provide an experimentally validated threedimensional model of the binding site in hSERT for the SSRI (S)-citalopram (Lexapro) using mutational analysis of hSERT paired with structure-activity data for (S)-citalopram analogs. LeuT structures are used to create homology models of hSERT, followed by docking of (S)-citalopram. Validation of binding models was performed based on the mutational dataset from 64 hSERT point mutants ...

Background: SERT and NET are important targets for antidepressants. Results: Antidepressants are differentially affected by mutations within the central S1 pocket of SERT and NET. Conclusion: Our data indicate that many antidepressants bind within the S1 pocket, and inhibitor selectivity is determined by residues within this site. Significance: This study provides a framework for modeling of drug binding, which may be used in future structure-based drug design.

G proteins are key mediators of G protein-coupled receptor signalling, which facilitates a plethora of important physiological processes. The cyclic depsipeptides YM-254890 and FR900359 are the only known specific inhibitors of the Gq subfamily of G proteins; however, no synthetic route has been reported previously for these complex natural products and they are not easily isolated from natural sources. Here we report the first total synthesis of YM-254890 and FR900359, as well as of two known analogues, YM-385780 and YM-385781. The versatility of the synthetic approach also enabled the design and synthesis of ten analogues, which provided the first structure–activity relationship study for this class of compounds. Pharmacological characterization of all the compounds at Gq-, Gi- and Gs-mediated signalling provided succinct information on the structural requirements for inhibition, and demonstrated that both YM-254890 and FR900359 are highly potent inhibitors of Gq signalling, with FR900359 being the most potent. These natural products and their analogues represent unique tools for explorative studies of G protein inhibition.