Aptamers as positive modulators

What to do when you want to validate an assay for drug discovery, but there are little or no literature tools available?

Well according to a publication from a group at Astra Zeneca, the use of Aptamers could be one way to solve this problem. Aptamers are lengths of DNA or RNA, generally 20-100 bases, which could be used in the same manner as small molecule tools, binding to targets of interest.

Using the historically difficult target glycine receptor as a model system (GlyRα1), the authors  generated aptamer libraries of RNA using the SELEX methodology (systematic evolution of ligands via exponential enrichment). This process involves the exposure of an aptamer library to your target, which has been immobilised (here the team used a biotin/streptavidin interaction), any unbound material is washed away and bound material is collected and amplified by RT-PCR. This is repeated across a number of iterations, improving the overall success rate.

In this case the SELEX process was run with a variety of different sources of the glycine receptor.  The active molecules generated from this method were then further validated using a radioligand filter binding assay. This resulted in eight aptamers being selected for scale up and further profiling in a variety of glycine receptor assays.

Selective binding of the aptamers was shown using SPR using immobilized glycine receptor, this was further supported by immunofluorescence of fixed cells and live cell imaging experiments.  Functional profiling of the aptamers occurred using a membrane potential dye assay supported with patch clamp electrophysiology.

The SPR measurements revealed all eight aptamers had Kd values in the low nanomolar range.  Interestingly two of the eight aptameters had slow on and off rates of binding .The cellular locations from the imaging experiments showed that the majority of the aptamer was present in the cytosol, and to a lesser extent at the plasma membrane. The authors suggested the cytosol accumulation may be due to interactions with Golgi and endoplasmic reticulum.

The functional assays highlighted some interesting findings. Using the membrane potential dye assay, five of the aptamers gave results suggesting they were positive modulators of the glycine receptor.  When this was further explored with one of the aptamers (c2) in a single cell patch clamp it was shown to be a positive modulator.

Overall the publication uses a variety of different supporting techniques to identify a positive modulator aptamer of the glycine receptor.

Could these molecules have a brighter future than just tools and become an alternative to small molecule therapeutics?  The issue of stability and delivery of the treatment have to be solved in each case, but the answer is yes.  As the authors point out, the FDA has approved the aptamer Macugen used for the treatment of age related macular degeneration.  The significant drawback of this medication is the requirement that it has to be injected into the eye of the patient.  So for the moment, tools seem to the current use for aptamers, however other clinical uses will be developed.


Figure showing the positive modulation of glycine receptor using aptamer C2

Shalaly, N. D., Aneiros, E., Blank, M., Mueller, J., Nyman, E., Blind, M., Dabrowski, M. A., et al. (2015). Positive Modulation of the Glycine Receptor by Means of Glycine Receptor-Binding Aptamers. Journal of Biomolecular Screening, 20(9), 1112-1123. Retrieved from http://jbx.sagepub.com/cgi/doi/10.1177/1087057115590575


Blog written by Gareth Williams

Invertebrate models for CNS drug discovery

The blood brain barrier (BBB) provides an added challenge to successful neuroscience drug discovery programs. The presence of metabolising enzymes, tight junctions and efflux transporters in the BBB are effective at preventing the passage of xenobiotics into the central nervous system (CNS).

In response to this extra barrier, a variety of in silico, in vivo and in vitro models have been generated to try to understand and assess BBB permeability at earlier stages in the drug discovery process reviewed in depth by Abbott.[1] In general the majority in vitro models lack the structural and functional complexity of the BBB and require multiple assays for an adequate prediction of permeability. In vivo models on the other hand are reflective of the real situation of the BBB, but unfortunately are both low through put and resource intensive, making them inadequate for screening larger sets of compounds needed at the early stages of a drug discovery project.

This year I attended a conference (ISNTD-d3 2015), where I saw a presentation by Dr Peter Nielsen from the company N2MO, on an ex vivo insect brain model for assessing BBB permeability. (Presentation found here: http://www.isntd.org/#/isntd-d-2015-nielsen/4589881601). The model itself provides medium throughput (~6 compounds a day) method of assessing BBB permeability of a compound. The advantage this model provides is that the insect BBB is similar in structure to the mammalian BBB. Tight junctions, high content of lipids and similar ABC and SLC transporters are all present in the insect brain,[2] suggesting reasons for why the model correlates well to rat perfusion models, where in vitro models fail. The model also provides the opportunity to measure the kinetics of permeability, as well as generating a figure of total uptake, paracellular diffusion and transcellular transport can also be discriminated.[3] Overall the extent of data generated from this model provides an adequate platform for the early assessment of BBB permeability, a bonus considering that these assays can be run with the first research batch of compound as requirement of material is low.[4] A model well worth considering for neuroscience drug discovery projects.

N2MO company website – http://n2mo.co/

Blog written by Ryan West

[1]         N. J. Abbott, Drug Discov. Today Technol. 2004, 407–416.

[2]         S. Al-qadi, M. Schiøtt, S. Honoré, P. Aadal, L. Badolo, BBA – Gen. Subj. 2015, 1850, 2439–2451.

[3]         K. Hellman, P. A. Nielsen, L. R. Olsen, R. Verdonck, N. J. Abbott, J. Vanden Broeck, G. Andersson, Pharmacol. Res. Perspect. 2014, 2, 1–12.

[4]         P. A. Nielsen, O. Andersson, S. Honore, G. Andersson, Drug Discov. Today 2011, 16, 12–15.

Organs on a Chip – The next step in 3D culture

Methods of modelling complex diseases have developed dramatically in recent years. Co-cultures as well as 3D cultures are now widely used in the drug discovery process, but with advances in microfluidics more complex co-culture systems are being developed that permit the investigation of complex biological processes. In these models biomimetic devices are used that are engineered to represent the structural and functional units found in organs such as bone, heart, liver, lung, intestine, brain and kidney. Also engineered within these devices is the ability to assay the result, therefore these devices are transparent enabling visualisation of the cells and immunological staining as well as being able to sample the media for assaying secreted factors (2).

Device for studying interaction of neuronal and astrocytes by metabolic communication (3)

Device for studying interaction of neuronal and astrocytes by metabolic communication (3)

One example of this is a model from Kunze et al., (3) where they used such a device for an astrocyte neuronal co-culture. Astrocyte neuronal co-cultures are not new, but these typically use an astrocyte feeder layer and therefore measure physical interactions between neuronal cells and astrocytes. In this model however, the group wanted to measure the effect of non-physical interactions between these cell types, i.e. secreted factors, or metabolic communication as described by the group. This microfluidic device is designed with two side channel in which astrocytes and neurons are seeded, separated by a central ‘contact’ area. The contact area in the middle enables fluid movement between the two cell types and neurite outgrowth, but the distance of 0.9mm means there is no physical contact between the cell types. Using this set-up the team was able to observe that astrocytes stably expressing mutant SOD (super-oxide dismutase) reduce neuronal survival in the ‘contact area’ compared to when the astrocytes express wild-type SOD, with no physical interaction between the astrocytes and neurons. Although this was a case-study for the device it demonstrates that this system can clearly be used for compounds targeting non-physical cross-talk between astrocytes and neurons as well as other cell-types. Additionally read-outs using both immunological staining of neurons or astrocytes as well looking at factors secreted can be used demonstrating the flexibility of the system.

Another example is a model that enables endothelial function to be observed in a 3D microenvironment (1). Here, a microfluidic device has an inner channel that in the study was lined with HUVECs (human umbilical vein endothelial cells). Inside the channel was a matrix gel where the group cultured A549 lung cancer spheroids. The device itself was clear and therefore enabled direct visualisation of the spheroids in the channel, therefore the group was able to measure EMT (Epithelial-mesenchymal transition)-induced spheroid dispersal due to the interaction of the HUVECs and the A549s. A number of FDA approved compounds known to inhibit EMT were applied to the inner matrix and their ability to inhibit EMT measured. Interestingly these compounds were as much as three fold more potent in the 3D assay than the conventional 2D equivalent. Interestingly the efficacious concentrations identified in the 3D micro-device were more similar to those measured in human trials than those in the 2D model.

To study epithelial-mesenchymal transition A549 spheroids are cultured in a gel-filled inner channel lined with HUVECs (1)

To study epithelial-mesenchymal transition A549 spheroids are cultured in a gel-filled inner channel lined with HUVECs (1)

These are simply two examples of sophisticated devices capable of modelling diseases or cell interactions that normal 2D or even 3D culture is unable to do. As the accessibility of microfluidics is expanding, so are the number and complexity of these devices. The limitations are clear; current throughput is low, labour intensive, and cost is high in comparison to traditionally screening. They do, however, provide possibilities for use in drug discovery such as late stage screening as well as target validation.


  1. Aref AR, Huang RY-J, Yu W, Chua K-N, Sun W, Tu T-Y, Bai J, Sim W-J, Zervantonakis IK, Thiery JP, Kamm RD. Screening therapeutic EMT blocking agents in a three-dimensional microenvironment. Integr. Biol. (Camb). 5: 381–9, 2013.
  2. Esch EW, Bahinski A, Huh D. Organs-on-chips at the frontiers of drug discovery. Nat. Rev. Drug Discov. 14: 248–60, 2015.
  3. Kunze A, Lengacher S, Dirren E, Aebischer P, Magistretti PJ, Renaud P. Astrocyte-neuron co-culture on microchips based on the model of SOD mutation to mimic ALS. Integr. Biol. (Camb). 5: 964–75, 2013.

Blog written by Trevor Askwith

A comparison of in vitro cellular viability assays

In the hunt for new drugs it is particularly important to understand if your compounds could have a negative impact on cellular health, even if they do interact with your target of interest. This requirement is determined with a variety of different measures and steps within the drug discovery process. One early and quick method is the use of in vitro cellular viability assays.

A group from the University of Otago compared some of the different in vitro cellular viability assays available in the following recent publication (Single, A. et al J Biomol Screen 2015, in press).

The authors wanted to investigate any differences between the more conventional viability endpoint measurements, such as resazurin based (a fluorescent based measurement) and CellTiter glo® (a luminescent based assay) with nuclear counting techniques (fluorescence based imaging technique). Further comparisons were made with the more recent developments from xCELLigence (an electrode impedance measurement) and IncuCyte (live cell imaging) assays. Both these latter methods can measure in kinetic intervals, which may offer a different insight by measuring cell growth rates in response to treatment with different compounds.

All the assay methods were tested using the MCF10A cell line and additionally the CDH1-negative isogenic line for which the compound vorinostat would be synthetically lethal. Taxol was used as a non specific toxic compound control.

Figure1-Gareth 28-09-15

First the endpoint assays were compared, the nuclear counting method showed a greater detection of reduced viability compared to the resazurin and CellTiter glo® methods. Another point of interest was that the CellTiter glo® showed the lowest sensitivity of all three measures. This was quite surprising given my experience of the use of this assay format and as it is a widely quoted assay in published literature. It would be interesting to see if the same trend occurs in other cell lines or if it was specific to this cell type. Also, a wider panel of compounds would be a useful adjunct to this work.
The Kinetic based platforms (the IncuCyte and xCELLigence) were used to determine proliferation rate during the logarithmic growth phase of the cell lines and also at full confluence of the cells. During the log phase, both systems reported reduced growth rate matching the different dose additions of the compounds. For the xCELLigence system slower rates were achieved, but the authors suggested this could be due to lower cellular adhesion of this cell type and thereby reducing the number of cells being detected. Once the cells had reached full confluence, the difference between treatment and non treatment of compound became very small, so it appears the use of these kinetic based platforms requires measurements in the log phase to generate the most reliable data.
As the group wanted to develop a method to identify synthetically lethal compounds against this cell line, they further investigated the use of a multiplexed assay format to overcome the limitations of the different methods they encountered. They combined both the resazurin and nuclei counting methods with the IncuCyte measurement and determined viability ratios with the results.
Using the multiplexed format the authors could determine the synthetically lethal effect of the vorinostat at both the log phase and full confluency of the cells. It was also noted that certain Taxol concentrations produced reduced viabilities during the growth phase of cells, but when full confluence endpoint was measured the cells had recovered to generate the similar readings as DMSO controls. This again highlights the advantage of using a kinetic measurement to discern these very subtle differences.
In summary the authors suggested the use of the multiplexed assay format as the most sensitive way to determine synthetically lethal compounds, but if that was not possible, they suggest the use of nuclear counting as it appeared to generate the most sensitive endpoint result. Overall, this was an interesting publication, which has challenged some of my preconceptions about the most optimal assay to use for this type of experiment. It is also interesting to see these more recently developed methods in real world action.

Blog written by Gareth Williams

Aggregation false positives in cell based assays?

An article dealing with the common problem of compounds that are false positives in screening assays was published recently (http://www.ncbi.nlm.nih.gov/pubmed/23437772).  One cause of compounds acting as false positives in screening assays is that they can self-aggregate, forming colloidal particles. This aggregation effectively sequesters the protein from its target and prevents activity. This has been a common problem in drug screening assays, particularly with soluble protein methods. In this publication the Shoichet lab at University of California, and others, have been investigating a further scope of the problem by examining  GPCR assays using a cell based format.

They took four compounds that were known to form aggregates and measured the activity against a variety of receptors using the Beta- Arrestin assay. The results show that these compounds were acting as antagonists against the receptors when they were stimulated with their agonist ligand, and this activity could be reversed with the addition of detergent or the use of centrifugation.

They also observed inverse agonism when the compounds were tested against the receptor in the absence of the activating ligand of the receptor, maybe via membrane perturbation.

It all highlights a type of assay artefact, which was thought to be more prevalent in soluble protein assays, can also have a bearing in cell based formats.The steps show by the authors (centrifugation and detergent usage) should be included to reduce the chance of false positives even if you are using a cell based method.

gw1Figure extracted from: Sassano, M. F., Doak, A. K., Roth, B. L., & Shoichet, B. K. (2013). Colloidal aggregation causes inhibition of g protein-coupled receptors. Journal of medicinal chemistry, 56(6), 2406–14. doi:10.1021/jm301749y