article

The role of drug transporters in phenotypic screening

The relative failure of molecular target-based drug discovery has led to a return to phenotypic screening. Targets that are intracellular necessitate their drug ligands to pass through plasma membranes, where the protein:lipid ratio is often 3:1 by mass and at least 1:1 by area. The widespread view that most of such transmembrane drug transport occurs through the phospholipid bilayer portion is incorrect: drugs must and do exploit transmembrane protein transporters that have been selected or evolved for the uptake of intermediary metabolites and natural products.

In some cases, the potency of extracellular drugs depends more or less exclusively on the activity of the transporters necessary to get them to their targets. Such considerations can explain the heterogeneous nature of drug efficacy and toxicity, and hence their roles in attrition, which highlights the need to consider drug transporters as a core component of the pipeline in both target-based and phenotypic drug discovery.

Following the basic recognition in chemical pharmacology that drugs must, in fact, have receptors,1 there arose two fundamental approaches to drug discovery. These can be referred to as phenotypic (function-first) and hypothesis-driven (gene- or target-based) (Figure 1). They are equivalent to the data-driven versus hypothesis-dependent approaches to science2 (Figure 1A), or to what are equivalently termed forward and reverse (chemical) genetics/genomics3,4 (Figure 1B). They can also be seen to differ in terms of the intellectual requirements of pre- and post-genomic research.5 Such diagrams (Figure 1) at once recognise two aspects: (i) that in a standard experimental programme there is (or should be) an iterative interplay between the gathering of data, their interpretation, and the design of follow-up experiments; and (ii) that different programmes can and do give dramatically different emphases to the two arcs represented in Figure 1B.

Figure 1A screening

Figure 1A: The cycle of knowledge

Classical drug discovery was entirely phenotypic: substances would have bioeffects that cured diseases, and therapies could be developed and tested (often then in whole animals) without any knowledge of their mechanisms; such knowledge, if required, could come later. By contrast, the advent of molecular cloning, recombinant protein production and (possibly surprisingly) genomics itself led to the desire to find molecular ‘targets’ whose activity could be assayed and modulated using very high-throughput screens in vitro.

Figure 1B screening

Figure 1B: Forward and reverse (chemical) genomics and drug discovery

Of course (Figure 1B) this would tell you that a particular molecule was highly potent at binding to and affecting (usually inhibiting) such a protein (receptor) but equally could tell nothing of whether off-target effects could occur (as they inevitably do6) and – more pertinently here – whether the drug might be able to reach an appropriate compartment such that it might have access to its targets, let alone whether it might also be toxic.

Figure 2 screening

Figure 2: Orally active drugs crossing epithelial cell layers

Specifically, orally active drugs must necessarily cross epithelial cell layers and any drugs with intracellular binding targets must necessarily enter cells. Figure 2 gives a high-level illustration of these features, lumping together all the import transporters (E1), drug metabolising enzymes (E2), and the efflux transporters for the drug (E3) and metabolites (E4). Binding sites (T, targets) for the free drug, as well as metabolisers and effluxers, lower its intracellular concentration and hence its availability. Many textbooks and reviews7-11 imply that there is an alternative (and indeed major) pathway of transmembrane drug transport that is often (misleadingly)12,13 referred to as ‘passive’ and is said to occur by simple diffusion through the phospholipid bilayer, mainly as a function of lipophilicity as assessed via log P or log D. Perhaps surprisingly, this is simply an assumption without any concrete evidence in intact cells, as the involvement of any such bilayer-mediated pathway has never actually been measured directly in real biological cells12,14 (and it would be almost impossible to do so). Years of evolution have almost certainly ensured that cells do not, in fact, tend to take up potentially toxic xenobiotics in such a way at substantial rates. Consequently, the evidence is, in our view,3,12-22 overwhelmingly to the effect that the transmembrane transport of pharmaceutical drugs occurs essentially exclusively via protein transporters (SLCs23), that have been selected during natural evolution for the transport of both intermediary metabolites24-27 and natural products.28,29 The detailed and extensive evidence is given in the papers cited,3,12-22 but the recognition that ‘phospholipid bilayer transport is negligible’ (PBIN)12 in intact biological cells clearly accounts for the very heterogeneous uptake of drugs in different cells,30 tissues, individual organisms and species (including or otherwise via the blood-brain barrier). The same principles (bilayer transport is negligible and transmembrane transport is transporter-mediated only) can be exploited for drug targeting.31-34 Consequently, no such phospholipid bilayer transport is considered to occur in intact biological cells, and is therefore not shown in Figure 2. In addition, there are many drugs that are known to have transporters whose molecular identity has not yet been uncovered: the natural product cocaine35 and the antipsychotic clozapine36 are just two examples.

Attrition

Attrition37-41 is a term used to describe the fact that even starting from phase 1 (‘first into humans’) presently some 92 percent of drug candidates fail to make it to market. This is of course extraordinarily costly. It has not improved since the move from classical to target-based strategies, and frankly, it is highly unlikely to – at least until the number of targets increases significantly as per systems pharmacology. The reasons are multiple and complex;38-44 mainly lack of efficacy or palpable toxicity. In our view, chief among them is the failure to recognise that the demonstrably heterogeneous distribution of drug transporters in different cells and tissues means that some tissues may have no uptake transporters (hence lack of efficacy) or they may have all too much of such activity and accumulate the drug to high levels (hence toxicity).

Figure 3 screening

Figure 3: Heterogeneous distributions of a drug in an organ can lead to a lack of efficacy while retaining the same gross PK/PD

For fundamental reasons,45,46 bulk measurements at the level of the organ or tissue cannot detect or discriminate this, a point illustrated in Figure 3. Importantly, phenotypic screening overcomes all of this, in the sense that one can at least hope to detect both efficacy and lack of toxicity in something closer to in vivo conditions – including the need for transmembrane transport – and consequently most successful drugs nowadays are discovered via early phenotypic screening.3,47-51

SLCs – the family of human SoLuteCarriers

Some 10 percent of the human genome encodes transporters and related proteins; they are the second largest component of the membrane proteome52 and an area of emerging excitement.53 Hediger and colleagues have helpfully systematised the taxonomy of human transporters;23 at the beginning of 2018 there were some 52 families,30 while the present total (maintained at http://www.bioparadigms.org/slc/intro.htm) is 65. These encode over 400 proteins. In addition, there are some 48 ABC- (ATP-binding cassette-) type transporters, mainly involved in efflux.54

The role of transporters in phenotypic screening

Space limitations mean that we must be selective in our illustrations, but because carrier-mediated transport is necessarily performed by genetically encoded protein transporters it is possible to modify the activity of named transporters by genetic or pharmacological means and observe the effects of such modifications, both on the consequent uptake of drugs and on their effects. This uptake may be measured directly (eg, using radioisotopes, fluorimetrically,55,56 or by mass spectrometry) but may also be inferred by the consequent bioactive effects of drugs that are taken up. The most extreme such effect, pertinent for instance in anti-cancer drug discovery, is cell death. The idea,57 then, is that cells that lack the transporters for toxic substrates are more resistant to those substances, and can be selected accordingly in competition experiments; analysis of the ‘winners’ can thereby indicate the relevant transporters. Transporters themselves can of course also be drug targets,58,59 although this is not a focus of the present review.

The modern availability of systematic collections of genetically modified strains (and the ease of their creation) makes such studies relatively straightforward.60,61 Thus, we found concentrations of drugs that lowered the growth rate of yeast cells by 90 percent and used57 the yeast knockout collection62,63 to assess those cells that were more resistant to cytotoxic drugs than were the wild-type strains. Typically (18/26 drugs tested), we could find between one and six knockouts that were more resistant to the drug than was the wild-type, although there were eight occasions when no such knockout had this effect. The interpretation of the latter is that when there are a great many possible uptake transporters the removal of just one does not result in an obviously sensitive phenotype. This is as expected given the theory of metabolic control analysis. 64,65

Figure 4A screening

Figure 4A: Differential transporter expression in cell lines

A particularly good example in the context of drug discovery came from a set of comparable experiments in mammalian cells reported by Superti-Furga and colleagues.66 They used a (near-)haploid cell line plus a ‘gene trap’ (retrovirus) that can insert itself into any gene.67 When such cells were exposed to normally lethal concentrations of the candidate anticancer drug sepantronium bromide (YM155), surviving cells were always found to have the retrovirus inserted into a gene encoding the solute carrier SLC35F2 (its ‘natural’ substrate is unknown). Such cells typically required 500-fold greater concentrations of YM155 to exhibit the same levels of toxicity, implying that at most 0.5 percent of the transmembrane flux could go via other routes (including the bilayer), which we certainly regard as negligible. Indeed, when studied in a series of cell lines, the toxicity varied over four orders of magnitude, in line with the expression level of the transcript for SLC35F2.66

Figure 4B screening

Figure 4B: Differential transporter expression in tissues

In a recent study,30 based on certain data,68 we assessed the variability of transporter expression in some 59 tissues and 56 cell lines; transporter expression levels typically varied to a significantly greater degree than did those of most other protein types,30 almost always by 100-fold and often by 10,000 times. We illustrate this in Figure 4 by showing the covariation of the distribution of two transporters (SLCO1B1, the main ‘statin’ transporter,69 and SLC22A4, a transporter for ergothioneine70,71) among 56 cell lines (Figure 4A) and 589 tissues (Figure 4B), that serves to illustrate how very heterogeneous their expression levels can be, often in cells from the same kinds of tissue. Within a tissue, methods such as imaging mass spectrometry show72 that they can be even greater. Data such as those in the present and previous paragraph indicate that predicting the phenotypic activity (toxicity) of drugs such as YM155 in the absence of knowledge of the transporters involved and their expression levels would be a hazardous enterprise indeed. 

Conclusions

There is a widespread and erroneous belief that most drugs can enter and exit cells ‘passively’ (ie, equilibrate) via a phospholipid bilayer common to them all. Similarly, the word ‘passive’ has often been misused to conflate the two ideas – one thermodynamic (equilibrative rather than concentrative) and one mechanistic (bilayer versus transporter-mediated transport). In fact, the bilayer route is negligible (or there is no evidence for it in intact biological cells). Consequently, to assess the potency of drugs with intracellular targets in phenotypic screens, we need first to know the transporters that they use to gain entry to (and exit from) the cells, and then their expression levels and activities (just as in any other systems biology programme).73-75 A failure to do so makes it much harder to understand the highly varying potencies (or toxicities) of drugs in different cells. By contrast, such knowledge can be of immense value, not least in targeting specific drugs to specific tissues – something that offers great hope for improving the therapeutic indices of potentially toxic drugs.31

Acknowledgments

We thank the BBSRC for financial support (grant BB//P009042/1).

Biography

Douglas Kell screeningDouglas Kell is a former Chief Executive of BBSRC and has a CBE for services to science and research.  He is currently Research Chair in Bioanalytical Science at the University of Manchester and in December 2018 will take up the position of Chair in Systems Biology at the University of Liverpool.  He is also Associated Scientific Director at the Novo Nordisk Foundation Centre for Biosustainability at the Technical University of Denmark.

Douglas has been a pioneer in many areas of computational biology and experimental metabolomics, including the use of evolutionary, closed-loop methods for optimisation.  He also contributed to the discovery of the first bacterial cytokine, currently on trial as part of a vaccine against tuberculosis.

He has published over 400 scientific papers with >26,000 citations (H-index 84) in WoK and >46,000 citations (H-index 105) in Google Scholar.  

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