Sven Gottschalk

MRI probes targeting glutamate receptors in the brain: Antagonist-based approach.

Introduction

Receptor targeting is widely used for competitive binding in brain imaging using techniques such as positron emission tomography and optical imaging. It is also an ongoing debate whether antagonist- or agonist-based approaches are more beneficial. However, an application of competitive binding for brain functional magnetic resonance imaging (fMRI) has not yet been shown. Our intention is to develop antagonist-based glutamate “responsive” MRI contrast agents (CAs) to image glutamate fluctuations in specific brain regions associated with neural activity. We have chosen CAs that bind to the metabotropic glutamate-receptor subtype 5 (mGluR₅) [1]. Such molecules can bind both to neuronal postsynaptic receptors as well as to those expressed on astrocytes. They can therefore act both as “markers” of receptor density and as indicator of neuronal activation. For the latter, ideally, upon glutamate binding to the receptor (i.e. after glutamate release at the synapse) the CA will be released, hence leading to a reduction in image contrast, followed by a restoration of equilibrium and re-binding of the CA to the receptor. These events are believed to occur over a period of few seconds allowing data acquisition using modern fast MR-techniques[2].

Goal

The study aims on developing functional MRI methods that are not based on the BOLD signal. Such methods would allow opening up a complete new way of functional assessment of how the brain works.

Methods

We have designed and synthesized different prospective CAs derived from various potent mGluR₅-receptor antagonists (Figure 1, alkynes like MPEP, MTEP shown in green and dipyridyl/heterobiaryl amides shown in brown) coupled to DOTA-derived macrocyclic gadolinium chelates. The CAs were evaluated in cultured primary cortical rat astrocytes, expressing mGluR₅ (verified by immunofluorescence). MRI-measurements to examine the ability of the CAs for cellular labeling were done with a 3T human whole body scanner. Antagonistic potency of the CAs was assessed with a calcium fluorescence assay, by which glutamate induced intracellular calcium transients mediated by mGluR₅ were measured. Antagonistic activity of the CAs was calculated as changes in EC₅₀ of glutamate. Receptor binding was monitored for the dipyridyl derivatives, as these compounds have an inherent fluorescence that changes upon binding. Commercially available receptor membrane preparations containing recombinant human mGluR₅A were used for these experiments.

Initial Results

Two of the gadolinium complexes retained significant antagonistic activity, one in each structural class. For the alkyne-derivative Gd.L³, an about fourfold increase of the EC₅₀(glutamate) (100µM CA, 15min, P<0.001) was found while under similar conditions the cellular relaxation rate R_1,cell increased to 126% of control (100µM, 45 minutes incubation time, P<0.001, Figure 2). The CA Gd.L⁸ derived from dipyridyl amides increased the EC₅₀(glutamate) about threefold (p<0.001) and the R_1,cell to 115% (p<0.05, Figure 2). Fluorescence measurements of the latter CA showed enhanced emission upon binding to mGluR₅-membrane preparations. This effect was reversed when increasing concentrations of glutamate were added, consistent with the reversibility of CA-receptor binding.

Conclusions

Using primary rat astrocytes as cellular model system to investigate newly developed glutamate-responsive MRI contrast agents, we were able to identify two promising candidates. These CAs are based on the structures of antagonists to mGluR₅ and our studies establish the validity of the concept, by which it might be possible to use MRI for brain functional measurements employing competitive binding approaches.

Figure 1: Structures of mGluR₅ targeted MRI probes.

Figure 2: Cellular ¹H-MR relaxation rates R_1,cell in cell suspensions after treatment of primary astrocytes with 100µM of [Gd.DOTA] or [Gd.L^1-8] for 45 min.

 Figure 3: Representative T₁-weighted MR-images of 1x10⁷ cells treated for 45 min with 100µM [Gd.DOTA] or [Gd.L²].

[1] Mishra, A., Gottschalk, S., Engelmann, J., Parker, D., Chemical Science 3(1)(2012)131-135

[2] Logothetis, N.K., Nature 453(2008)869-878

Application of CyLoP-1 for the induction of apoptosis in cancer cells.

Introduction

Cell penetrating peptides (CPPs) are known as transduction vectors for several years and their feasibility has been demonstrated in a wide range of in vitro studies [1]. Due to their ability to deliver a variety of compounds through cellular membranes they have also gained attention in the biomedical field. Unfortunately, uptake via endocytotic pathways and hence vesicular encapsulation limits the application of CPPs, particularly when it comes to a cargo that needs to be delivered to the cytosol of a cell. We have recently developed the new cysteine-rich CPP CyLoP-1 (Figure 1) [2]. It could be shown that the cysteines were essential for efficient uptake as well as cytosolic distribution and it was also proved that CyLoP-1 is taken up most efficiently in its natural L-form. To verify the capability of CyLoP-1 to transport a biological active cargo across cellular membranes, we attached the pro-apoptotic peptide-sequence AVPIAQK (SmacN7) to CyLoP-1. SmacN7 is derived from the N-terminus of the second mitochondria-derived activator of caspase (Smac) protein which is released from mitochondria in response to apoptotic stimuli and promotes caspase-3 activation in the cytosol [3]. The short sequence SmacN7 has been shown to be sufficient enough to maintain the original function of Smac [4]. However, SmacN7 alone is not capable of passing through cellular membranes and it needs to bind to its cytosolic target protein to exert its pro-apoptotic action [3, 4]. By measuring the apoptotic level in treated cells, SmacN7 can thus be used to prove the cytosolic delivery of CPP-SmacN7 conjugates [5].

Goal

The project aims on demonstrating the potential capability of CyLoP-1 for targeted cytosolic delivery of drugs or other compounds that are unable of crossing cellular membranes on their own.

Methods

CyLoP-1 (CRWRWKCCKK, Figure 1) was synthesized by Fmoc solid-phase synthesis. An additional lysine was attached as linker. Fluorescein isothiocyanate (FITC) as fluorophore was coupled to the ?-amino group of this additional lysine. At the ?-amino group of the same lysine SmacN7 (AVPIAQK) was covalently conjugated, yielding the construct SmacN7-K(FITC)-CyLoP-1. Internalization was evaluated on NIH3T3 mouse fibroblasts [2]. Briefly, confluent cells were incubated for 18 h with 2.5 µM of CPP-conjugate. Then, nuclei were counter-stained with Hoechst 33342 and extracellular fluorescence was quenched with trypan blue. Intracellular FITC- and Hoechst-fluorescence were measured in a plate reader and microscopic images of the same cells were taken afterwards.

Apoptosis was induced in confluently grown human cancer cell line (HeLa) with the combination of an agonistic anti-Fas antibody and cycloheximide (CHX) [5]. Incubations were done for 18 hours with or without SmacN7-K(FITC)-CyLoP-1 (2.5 or 5 µM). The extent of apoptosis was also assessed after 18 hours of treatment with the SmacN7-CyLoP-1 construct alone. Caspase-3 activity as marker for the extent of apoptosis was measured in the supernatant of lysed cells with a standard enzymatic assay. The caspase-3 activity measurements were normalized on the protein content of the samples which was quantified with a standard Bradford protein assay.

Initial Results

Treatment of HeLa cells with only SmacN7-K(FITC)-CyLoP-1 induced already a significant and concentration dependent increase of caspase-3 activity (Figure 2). However, this increase was on a lower absolute level as compared to apoptosis induced by CHX+anti-Fas-treatment (the caspase-3 activities were 0.4±0.1 and 4.7±0.8 [DFU sec^-1] for untreated control and CHX+anti-Fas, respectively). Furthermore, co-treatment of HeLa cells with CHX, anti-Fas antibody and the SmacN7-CyLoP-1 construct enhanced the level of apoptosis in addition to that already induced by CHX+anti-Fas-treatment alone (Figure 3).

Conclusions

We have shown that our newly developed cysteine-rich peptide CyLoP-1 is superior in reaching the cytosol of cells in comparison to other established CPPs. We have also demonstrated that the cargo construct SmacN7-CyLoP-1 was capable of inducing and enhancing apoptosis in cancer cells, thus proving the ability of CyLoP-1 to successfully deliver a fully functional attached bioactive cargo to its cytosolic site of action.

Figure 1: Schematic structure of a CyLoP-1 cargo conjugate.

Figure 2: Induction of apoptosis; Caspase-3 activity 18 h after incubation with SmacN7-K(FITC)-CyLoP-1.

Figure 3: Enhancement of apoptosis; Caspase-3 activity 18 h after incubation with CHX, anti-Fas and SmacN7-K(FITC)-CyLoP-1.

References:

[1] Lundberg et al., J. Mol. Recognit. 16(2003)227.

[2] Jha D, Mishra R, Gottschalk S, Wiesmüller KH, Ugurbil K, Maier ME, Engelmann J. Bioconjug Chem, 22(2011)319.

[3] Fandy et al., Mol. Cancer 7(2008)60.

[4] Heckl et al., Med. Chem. 4(2008)348.

[5] Duchardt et al., Traffic 8(2007)848.