The roles of transporters in the human metabolic network

When you eat a foodstuff or a pill (i.e. a pharmaceutical drug) it is important that the relevant molecules go to the places where they will be of most benefit. How nutrients and drugs are absorbed and distributed (and eventually excreted) is thus a topic of high importance. When it goes wrong in the case of drugs they may not work properly or may even be toxic; this latter is known as an Adverse Drug Reaction, and they account for more than 5% of hospital admissions. Cells are bounded by cell membranes, whose job it is to stop them letting in any old rubbish. Instead, these membranes contain proteins called transporters that serve to ferry small molecules into and out of cells as part of the day-to-day reactions (metabolism) that keep us alive. Such transporters account for fully one third of the gene products involved in these biochemical networks. It turns out in general terms that, since all they recognise is a molecule, without knowing its 'purpose' (nutrient, drug, vitamin, etc), just these same transporters are involved in transporting nutrients, vitamins and pharmaceutical drugs into cells; the problem is that we do not tend to know which transporters transport which substances, and this correlation-based assessment is what we wish to find out here.

To do this we shall study how transporter levels and the extent of small molecule uptake vary together between different cells and tissues; given enough of these paired measurements we can work out which changes in which transporters best account for the changes in small molecule uptake, including molecules not used in the learning phase, and can then test directly that they do indeed transport the molecules we claim.

Because the transporter levels naturally vary between tissues they must naturally be controlled, and several substances (such as vitamin D) are known to affect these levels dramatically. This means that we can expect to be able to modulate the expression of transporters and different tissues by adding a second molecule (a so-called 'binary weapon'), and thereby 'force' particular substances to go only to those tissues. We shall test this by adding a library of small artificial (man-made) molecules (so-called 'fragments') and seeing which of them can do this (at least to some degree). Computer methods help us to find larger and more potent molecules that possess these same fragment signatures and that would therefore be predicted to have the desired targeting effects.

Importantly, we shall curate all of the data in a web-accessible database.

Based in part on our reconstruction of human metabolism, where fully one third of the gene products in the human metabolic network are transporters, albeit usually of unknown specificity, we have argued, with considerable evidence, that these self-same transporters are responsible for moving all kinds of molecules, including nutrients and pharmaceutical drugs, into (and out of) cells. We now need to find which transporters are used by specific substances. The strategy is to study their covariation in a series of different cell lines and human tissues, where the availability of such paired data will easily admit the deconvolution of which transporter levels best explain the differential distributions of drugs between cells and tissues (or tissue subtypes). Modern SWATH-based proteomics will give the transporter levels, and similar MRM-based mass spectrometric methods will determine the drug concentrations. In addition, further variation of transporter and drug uptake levels will come from the use of the near-haploid KBM7 cell lines exposed to a suitable gene trap.

Because tissue expression profiles of transporters vary hugely between (and indeed within) tissues, something must be controlling them (we know that vitamin D is one such element). We can thus look for small molecules that affect these processes and thus cause the high and ideally sole expression of specific transporters in specific target cell types. We shall use a novel, fragment-based phenotypic approach to find such molecules. The result will be a first demonstration of the use of a second molecule to target a first molecule uniquely or predominantly to a particular target tissue. We shall here use antibiotics as our test system, as poor penetration is responsible in significant measure for the increasing antimicrobial resistance.

Importantly, we shall curate all of the data in a web-accessible database.

WHO WILL BENEFIT: Companies will benefit in a number of ways, by (i) gaining access to a database on the cell and tissue distributions of a variety of proteins of significant biological interest, (ii) the knowledge of methods used for the rapid detection and estimation of proteins using modern proteomics methods, and (iii) knowledge of the QSARs of the various transporters, whether the molecules are nutrients, intermediary metabolites, nutraceuticals, drugs, or bioactives in personal care products. This will also be important for use in the modelling of drug disposition by companies such as Simcyp (now part of Certara).

So far as pharmaceutical drug discovery is concerned, attrition rates, even from 'first into humans' are currently running at 92% (latest Tufts analysis; see http://lifescivc.com/2014/11/a-billion-here-a-billion-there-the-cost-of-making-a-drug-revisited/), taking the average cost of drug development (by simple arithmetic) from ca $200M to an eye-watering $2.5Bn. The attrition is caused mainly by lack of efficacy and by toxicity. If our views are correct, a chief beneficiary in time will be the pharmaceutical industry, as they will benefit from the ability to exploit drug transporters in targeting drugs to precise tissues. In effect we are challenging the current means of drug development because at present chemicals incapable of cell entry are being screened as drug candidates. The new strategy only intends to screen libraries of substances representing the chemical space capable of transporter interaction to enter cells - and does so at the onset of drug development.

HOW WILL THEY BENEFIT: As is our practice, all pertinent data are made available via the Web, and OA publishing has long been our norm. We also hold frequent workshops in Manchester to assist dissemination of research results. We have pioneered in the Altmetrics field for digital dissemination - indeed a recent Nature article (Kwok R: Altmetrics make their mark. Nature 2013; 500:491-492) highlighted the fact that the PI's paper Hull D, Pettifer SR, Kell DB: Defrosting the digital library: bibliographic tools for the next generation web. PLoS Comput Biol 2008; 4:e1000204 was the most accessed in ANY PLoS journal, with over 53,000 accesses! (it is past 95,000 now) - the PI's paper Kell DB: Iron behaving badly: inappropriate iron chelation as a major contributor to the aetiology of vascular and other progressive inflammatory and degenerative diseases. BMC Med Genom 2009; 2:2 has over 85,000 accesses, increasing at ~50 per day).We shall work closely with University KT staff and industrial IP offices (UMIP in Manchester) to secure intellectual property rights for any useful inventions that we discover. Having secured IP, future development work can take place, and several routes to commercialisation can be explored. For example, all pharmaceutical companies have their own relevant groups, with whom we are in contact. Finally, having secured IP, we shall, of course, seek actively to communicate our scientific findings to the wider research community through scientific meetings, scholarly publications and press releases.

THE WIDER COMMUNITY: DBK is also a well known blogger and tweeter, and social media will provide a novel and useful means of disseminating our findings.

COMMUNICATIONS: We will communicate with relevant industrial partners both directly and via the meetings of relevant learned societies (we are members of several). In year three of the Project, we will organise a half-day meeting to explain our research to interested industrial scientists. However, we will also provide a video link to facilitate the participation of those who are unable to travel to Manchester.