Nanoscience Meets Drug Discovery
By: Carol A. Rouzer, VICB Communications
Published: March 7, 2012
Using ligand-conjugated quantum dots provides an innovative way to screen for new drugs to treat major depression.
Major depression is a real cause of human suffering that affects between 2 and 5% of the U.S. population at a cost of more than $100 billion a year. Currently, the most frequently prescribed drugs for the treatment of depression are the selective serotonin (5-hydroxytryptamine, 5-HT) reuptake inhibitors (SSRIs), sold under the familiar brand names of Prozac, Paxil, Zoloft, Celexa, and Lexapro. 5-HT is a brain chemical that communicates signals related to mood, appetite, and feelings of well-being. Evidence suggests that, in some individuals, a deficiency of 5-HT signaling contributes to the risk for major depression. Following release at nerve endings, or terminals, 5-HT is inactivated by reuptake at the terminal via the 5-HT transporter (SERT). Because SSRIs block SERT-dependent 5-HT reuptake, they act to elevate 5-HT-mediated signaling, strengthening 5-HT’s actions (Figure 1).
Figure 1. Serotonin (5-HT) transmission at a synapse. 5-HT (pink triangles) is synthesized from tryptophan and stored in vesicles at the axon terminal. Upon stimulation, the vesicle fuses with the plasma membrane releasing the 5-HT into the synaptic cleft. Binding of 5-HT to specific receptors triggers a response in the dendrite on the other side of the synaptic cleft. The signal is terminated by reuptake of 5-HT by the 5-HT transporter (SERT, green). Blockade of SERT by SSRIs leads to a prolongation of 5-HT-mediated neurotransmission. Reproduced from the National Institute of Drug Abuse.
Despite their emergence as the therapy of choice for many depressed patients, SSRIs have the disadvantage of slow onset of action (up to six weeks to reach therapeutic benefit) and some unwanted side effects, such as sexual dysfunction and increased risk of suicide among younger patients. Thus, an ongoing effort exists to find better drugs. In one gambit, investigators are searching for multifunctional compounds that block the reuptake of 5-HT as well as other neurotransmitters (e.g. norepinephrine) known to also play a role in mood circuits. In a second line of research, investigators are searching for compounds that alter SERT activity by binding to sites on the protein other than the 5-HT binding site, so-called “allosteric inhibitors”. Agents that modulate SERT rather than blocking it completely could provide more effective and safer treatments. Unfortunately, the search for such medications is hindered by the lack of rapid, inexpensive, high-throughput screens that can identify SERT allosteric modulators. Vanderbilt Institute of Chemical Biology (VICB) members Sandra Rosenthal and Randy Blakely have sought to improve this situation through the development of a new high-throughput screen for SERT inhibition using ligand-conjugated quantum dots [J. C. Chang et al. (2011) J. Amer. Chem. Soc., 133, 17528].
Quantum dots are tiny particles (2 to 10 nm in diameter) composed of a semiconductor material (usually selenides or sulfides of zinc or cadmium). Due to their small size, they possess unique optical properties, specifically fluorescence in the visible spectrum. Because the wavelength of the fluorescence decreases with particle diameter, quantum dots can be “tuned” to fluoresce at any desired wavelength by controlling particle size. Compared to most fluorophores that are commonly used in bioassay design, quantum dots are more sensitive, and they do not “bleach” with prolonged illumination. Thus quantum dots offer numerous advantages for use in screens for the identification of new SERT inhibitors.
The Rosenthal and Blakely labs had already identified an indole-containing compound that acted as an antagonist with high affinity for human SERT (hSERT). Linking the indole nucleus to an alkyl chain increased hSERT inhibitory activity, possibly as a result of interactions between the hydrophobic chain and intramembrane regions of the protein. Addition of a polyethyleneglycol (PEG) chain to increase water solubility and a biotin group to enable streptavidin binding yielded the compound IDT318 (Figure 2).
Figure 2. Structure of IDT318 showing the indole nucleus (I), alkyl chain (II), polyethylene glycol chain (III), and biotin group (IV). Reproduced with permission from Chang et al. (2011) J. Amer. Chem. Soc., 133, 17528. Copyright 2011, American Chemical Society.
The investigators showed that incubation of frog oocytes expressing hSERT with IDT318 followed by addition of quantum dots bound to streptavidin (SA-QD) resulted in labeling of the oocyte plasma membranes with a red fluorescent glow (Figure 3-1). The fluorescence resulted from the formation of an IDT318/SA-QD complex bound to hSERT. When the same experiment was carried out using oocytes that did not express hSERT, no fluorescence was detected (Figure 3-2).
Figure 3. Fluorescent labeling of an hSERT-expressing oocyte (1) by incubating the cell first with IDT318 and then SA-QD. No labeling is observed when the oocyte does not express hSERT (2). Reproduced with permission from Chang et al. (2011) J. Amer. Chem. Soc., 133, 17528. Copyright 2011, American Chemical Society.
The investigators hypothesized that addition of a second compound that binds to hSERT would displace the IDT318/SA-QD complex either by directly competing for its binding site, or by binding to a separate allosteric site, leading to a reduction in affinity for the IDT318/SA-QD complex (Figure 4).
Figure 4. Competition for hSERT labeling by the IDT318/SA-QD complex can occur through direct binding of competitor to the same site on the protein (left), or binding of an allosteric modulator at a different site that reduces the binding affinity for the fluorescent complex (right). Reproduced with permission from Chang et al. (2011) J. Amer. Chem. Soc., 133, 17528. Copyright 2011, American Chemical Society.
Indeed, they found that addition of the hSERT antagonist paroxetine to IDT318/SA-QD-labeled oocytes resulted in a time- and concentration-dependent decrease in fluorescence (Figure 5). Since the IDT318-conjugated quantum dots were already bound to SERT before paroxetine was added, loss of fluorescence suggests that paroxetine is acting as an allosteric SERT modulator. Kinetics studies showed that, for the first ten minutes following paroxetine addition, oocyte fluorescence decreased by a first order process. The data readily yielded an apparent dissociation rate constant that increased, as expected, with paroxetine concentration. Loss of fluorescence upon incubation of labeled cells in the absence of a displacing ligand was less than 10%, indicating that fluorescence quenching and spontaneous ligand dissociation were not a significant problem.
Figure 5. Addition of paroxetine to IDT318/SA-QD-labeled oocytes results in a time-and concentration dependent loss of fluorescence (A). The quantitative data (B) plotted on a semi-log plot (C) reveal that the loss of fluorescence is a first-order process. Reproduced with permission from Chang et al. (2011) J. Amer. Chem. Soc., 133, 17528. Copyright 2011, American Chemical Society.
The investigators concluded that the displacement of IDT318/SA-QD complexes bound to hSERT-expressing oocytes displays the necessary sensitivity and rapidity to be used for the screening of new allosteric compounds in an antidepressant drug discovery program. As the method does not utilize expensive radiolabel techniques, it should be readily adaptable to a high-throughput format. These efforts represent the first attempt to devise a target-selective drug discovery platform using ligand-conjugated quantum dots for the discovery of allosteric modulators.