Evaluating Cellular Drug Uptake with Fluorescent Sensor Proteins

We are introducing a new approach to evaluate cellular uptake of drugs and drug candidates into living cells. The approach is based on converting the protein target of a given class of compounds into a fluorescent biosensor. By measuring the binding of different compounds to their cognate biosensor in live cells and comparing these values to those measured in vitro, their cellular uptake and concentrations can be ranked. We demonstrate that our strategy enables the evaluation of the cellular uptake into the cytosol of 2 classes of inhibitors using two different sensor designs; first, sensors comprising the self-labeling protein SNAP conjugated with a chemically modified inhibitor shown for inhibitors of the enzyme human carbonic anhydrase II; and a label-free sensor for inhibitors of protein−protein interactions demonstrated for the protein pair p53−HDM2.

Functional Characterization of the Mouse Olfactory Receptor mOR256-17 in Live Cells

Thamani Dahoun

Olfactory receptors (ORs) constitute the largest family of sensory membrane proteins in mammals. They play a key role within the olfactory system to recognize and discriminate a nearly unlimited number of structurally diverse odorant molecules. The molecular basis of OR-mediated odor signal detection and transduction is poorly understood. This is due in part to difficulties in functional expression of ORs at high yield, preventing structural and biophysical studies at the level of the receptor protein. Here we report on the heterologous expression of the mouse receptor mOR256-17 yielding an average of 106 ORs per cell in transiently transfected mammalian cells. For quantification and optimization of OR expression we employed different fluorescent tags. Green fluorescent protein fused to the C-terminus of mOR256-17 allowed quantification of total cellular OR biosynthesis, and posttranslational fluorescence labeling of a 12 amino acid polypeptide sequence at the N-terminus permitted the selective visualization and quantification of ORs at the plasma membrane using cell flow cytometry. The establishment of a mOR256-17-inducible HEK293S-TetO cell line allowed us to determine by fluorescence spectrofluorometry and fluorescence correlation spectroscopy (FCS) the optimal conditions to solubilize the receptor. By screening a large odorant compound library we discovered a new selective spectrum of potent mOR256-17 specific agonists. Furthermore, by using a reporter assay that couples both Gs and Gq proteins after inhibition of adenylate cyclase by the chemical adenylyl cyclase inhibitors 9-(tetrahydro-2-furanyl)-9H-purin-6amine (SQ22536) and after the inhibition of phospholipase C by the chemical inhibitor 1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), we identified one agonist which induced the inositol 1,4,5-trisphosphate (IP3) transduction pathway. We provided further evidence of the involvement of this second messenger (i) by employing another reporter gene specific to the Gq pathway and (ii) by using electrical impedance spectroscopy. Our attempt to create a FRET fluorophore pair system based on fluorescein arsenical hairpin (FlAsH) labeling and on carrier protein tags to study the receptor conformational changes during activation revealed that modifications in mOR256-17 sequences perturbs the correct receptor folding and prohibits its expression at the plasma membrane. Our study indicated that an essential prerequisite for achieving cell surface expression of mOR256-17 in heterologous cells is the co-expression of the accessory protein RTP1 or its short version RTP1S, which are also expressed by olfactory sensory neurons. Elsewhere, Saito and co-workers have proposed that this protein interacts with OR when the OR is expressed heterologously, but they have not provided evidence to prove the role of RTP1 towards OR. To investigate whether direct physical interactions between mOR256-17 and RTP1 occurs during receptor trafficking to the cell membrane, we labeled mOR256-17 and RTP1 orthogonally using the acyl carrier protein (ACP) and the peptidyl carrier protein (PCP) respectively. In addition, cyan fluorescent protein fused to the N-terminus of RTP1 enabled to study RTP1 biosynthesis in presence and absence of the mOR256-17; here we observed that a large portion of RTP1 remains in the endoplasmic reticulum while the mOR256-17 reaches the Golgi apparatus when it is co-expressed with RTP1S. Furthermore, by using two different RTP1S chimera, we could show that the cytosolic part of RTP1S is important for the mOR256-17 folding and trafficking.

EPFL2013

Spatio-temporal synchronization of biological oscillators

Jonathan Bieler

The circadian clock is a cell-autonomous and self-sustained oscillator with a period of about 24 hours that controls many aspects of cellular physiology. The cell cycle is also a fundamental periodic process, with a period in the range of one day in mammals. A large number of studies showed that these two cycles interact at the molecular level. Mathematical theory of coupled oscillators predicts that in the presence of coupling, two cycles with similar periods can synchronize. Consequently, the molecular interactions between the cell cycle and the circadian clock could lead to their synchronization. Such synchrony is consistent with a number of studies showing that cells divide more at specific circadian times. Moreover, a few works also report effects of cell divisions on the circadian oscillator. More detailed mathematical models of the molecular networks involved also predict that the two cycles can synchronize. To better characterize this potential synchronization in mammalian cells, we monitored the mutual interactions between the two oscillators by time-lapse imaging of single NIH3T3 fibroblasts during several days. To do so, we used a fluorescent reporter under the control of a circadian promoter and monitored the cell cycle progression by detecting the time of mitosis. The analysis of thousands of circadian cycles in dividing cells clearly indicated that both oscillators tick in a 1:1 mode-locked state, with cell divisions occurring tightly 5 hours before the peak in circadian Rev-Erbα -YFP reporter expression. In principle, such synchrony may be caused by coupling from one oscillator onto the other, or in both directions. While gating of cell division by the circadian cycle has been most studied, our data combined with stochastic modeling strongly suggest that reverse coupling is predominant in NIH3T3 cells. In order to further test the direction of the coupling, we performed several perturbation experiments; we changed the cell cycle period by acquiring recordings at different temperatures, we analyzed cell lines containing genetic knock-down of circadian genes and used drugs that target specific proteins of the cell cycle and the circadian clock. These experiments showed that the two interacting cellular oscillators adopt a synchronized state that is highly robust over a wide range of parameters. Moreover, the set of perturbations on the periods of either oscillator induced shifts in the relative phases of circadian and cell cycles that were consistent with a scenario in which the circadian cycle resonates to the cell cycle. These findings have implications for circadian function in proliferative tissues, including epidermis, immune cells, and cancer.

EPFL2014

Fluorescent sensor proteins to assess the cellular uptake of therapeutic peptides and small molecules

Silvia Scarabelli

The determination of cell membrane permeability and of the absorption and clearance rates is an important step in the pharmacokinetic profiling of a new drug candidate. In a similar way, research in cell biology relies on the development of cell penetrating biochemical probes allowing to study and interfere with cellular functions and physiology. A variety of methods have been developed to estimate the ability of molecules to enter the intracellular space. They are based either on artificial membranes or on cellular models, with consequently different abilities to mimic the cellular permeation mechanisms. Howev-er, these techniques are often laborious, expensive and have to be combined to get a reliable characterization of the properties of the molecule of interest. This thesis introduces a new strategy to evaluate real-time absorption and clearance kinet-ics of different classes of molecules in living cells. The approach is based on the use of in-tracellular single-chain FRET-based biosensors with a versatile modular design. First, a proof-of-concept bioprobe for inhibitors of the enzyme human carbonic anhydrase II (HCAII) was developed and optimized for intracellular use: the cell-entry dynamics of a selection of well-known sulfonamide drugs was compared and monitored in real time. Then, we adapted the design of the sensory protein to generate a label-free biosensor for inhibitors of the p53-HDM2 interaction, a highly studied protein-protein interaction in cancer biology. The per-meability and the kinetics of cell entry and washout of various underivatized small molecule and peptidic inhibitors could be measured. This thesis represents a step towards the introduction of a general approach for a low-cost, rapid and minimally invasive evaluation of molecular permeabilities in the cell type of choice. Their modular design lends itself to be adapted to the detection of other families of molecules, provided that a binding protein for the molecule of interest is known.

EPFL2017