The roles of transporters in the human metabolic network

Lead Research Organisation: University of Manchester
Department Name: Chemistry

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
publication icon
Sait A (2021) Viral Involvement in Alzheimer's Disease in ACS Chemical Neuroscience

publication icon
Darbani B (2018) Energetic evolution of cellular Transportomes in BMC Genomics

publication icon
Kell D B (2017) The role of drug transporters in pheotypic screening in Drug Target Review

publication icon
Kerley RN (2018) The potential therapeutic effects of ergothioneine in pre-eclampsia. in Free radical biology & medicine

 
Title How drugs and nutrients get into cells 
Description Commissioned animation, 3.23 mins long. 
Type Of Art Film/Video/Animation 
Year Produced 2019 
Impact The animation was advertised throughout the University of Liverpool using internal communications. It has also been brought to the attention of collaborators across the world. Seeking to understand how drugs and nutrients get into living cells is not how a lot of textbooks portray it. 
URL https://youtu.be/s23vNwLE-Jw
 
Description Novel housekeeping genes.
Exploitation Route Better normalisation
Sectors Pharmaceuticals and Medical Biotechnology

 
Description (ReSOLUTE) - Research empowerment on solute carriers (ReSOLUTE)
Amount € 23,850,000 (EUR)
Funding ID 777372 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 06/2018 
End 06/2023
 
Description REsolution - Add Medical Genetic Solutions to ReSolute
Amount € 1,000,000 (EUR)
Funding ID 101034439 
Organisation European Commission H2020 
Sector Public
Country Belgium
Start 05/2021 
End 12/2023
 
Description DTU 
Organisation Technical University of Denmark
Country Denmark 
Sector Academic/University 
PI Contribution Associated Scientific Director and Head of the Flux Optimisation and Bioanalytics Group The group's efforts seek to expand the uses of microbial cell factories through: Novel and high-throughput screening and methods for selection The development, identification and engineering of relevant membrane transporters Computational methods for making better strains The ultimate goal of the group's research is to contribute novel and robust bacterial cell factories for the efficient and sustainable production of fine biochemicals to satisfy society's needs.
Collaborator Contribution Collaboration with DTU academics and their research groups, funding to employ research associates and access to equipment and analytical facilities in Denmark, Manchester and Liverpool.
Impact A palette of fluorophores that are differentially accumulated by wild-type and mutant strains of Escherichia coli: surrogate ligands for profiling bacterial membrane transporters Salcedo-Sora, J. E., Jindal, S., O'Hagan, S. & Kell, D. B., 2021, In Microbiology. An untargeted metabolomics strategy to measure differences in metabolite uptake and excretion by mammalian cell lines Wright Muelas, M., Roberts, I., Mughal, F., O'Hagan, S., Day, P. J. & Kell, D. B., 2020, In : Metabolomics. 16, 10, 107. A quantitative survey of bacterial persistence in the presence of antibiotics: Towards antipersister antimicrobial discovery Salcedo-Sora, J. E. & Kell, D. B., 2020, In : Antibiotics. 9, 8, 36 p., 508. Automating cloning by natural transformation Jiang, X., Palazzotto, E., Wybraniec, E., Munro, L. J., Zhang, H., Kell, D., Weber, T. & Lee, S. Y., 2020, In : A C S Synthetic Biology. 9, 12, p. 3228-3235 8 p. Covid-19: The rollercoaster of fibrin(ogen), d-dimer, von willebrand factor, p-selectin and their interactions with endothelial cells, platelets and erythrocytes Grobler, C., Maphumulo, S. C., Grobbelaar, L. M., Bredenkamp, J. C., Laubscher, G. J., Lourens, P. J., Steenkamp, J., Kell, D. B. & Pretorius, E., 2020, In : International Journal of Molecular Sciences . 21, 14, 25 p., 5168. DeepGraphMolGen, a multi-objective, computational strategy for generating molecules with desirable properties: A graph convolution and reinforcement learning approach Khemchandani, Y., O'Hagan, S., Samanta, S., Swainston, N., Roberts, T. J., Bollegala, D. & Kell, D. B., 2020, In : Journal of Cheminformatics. 12, 1, 53. Deep learning and generative methods in cheminformatics and chemical biology: navigating small molecule space intelligently Kell, D. B., Samanta, S. & Swainston, N., 2020, In : Biochemical Journal. 477, 23, p. 4559-4580 Research output: Contribution to journal > Journal article > Research > peer-review Detection of Citrullinated Fibrin in Plasma Clots of Rheumatoid Arthritis Patients and Its Relation to Altered Structural Clot Properties, Disease-Related Inflammation and Prothrombotic Tendency Bezuidenhout, J. A., Venter, C., Roberts, T. J., Tarr, G., Kell, D. B. & Pretorius, E., 2020, In : Frontiers in Immunology. 11, 577523. Effect of L-Ergothioneine on the metabolic plasma profile of the RUPP rat model of preeclampsia Morillon, A. C., Williamson, R. D., Baker, P. N., Kell, D. B., Kenny, L. C., English, J. A., McCarthy, F. P. & McCarthy, C., 2020, In : PLOS ONE. 15, 3, e0230977. Engineered cells for production of indole-derivatives Kell, D., Yang, L. & Malla, S., 24 Sep 2020, IPC No. C07K 14/ 715 A I, Patent No. WO2020187739, 13 Mar 2020, Priority date 15 Mar 2019, Priority No. EP20190163184 Research output: Patent Erythrocyte, platelet, serum ferritin, and p-selectin pathophysiology implicated in severe hypercoagulation and vascular complications in COVID-19 Venter, C., Bezuidenhout, J. A., Laubscher, G. J., Lourens, P. J., Steenkamp, J., Kell, D. B. & Pretorius, E., 2020, In : International Journal of Molecular Sciences . 21, 21, 14 p., 8234. Gingipain R1 and Lipopolysaccharide From Porphyromonas gingivalis Have Major Effects on Blood Clot Morphology and Mechanics Nunes, J. M., Fillis, T., Page, M. J., Venter, C., Lancry, O., Kell, D. B., Windberger, U. & Pretorius, E., 2020, In : Frontiers in Immunology. 11, 1551. L-(+)-Ergothioneine Significantly Improves the Clinical Characteristics of Preeclampsia in the Reduced Uterine Perfusion Pressure Rat Model Williamson, R. D., McCarthy, F. P., Manna, S., Groarke, E., Kell, D. B., Kenny, L. C. & McCarthy, C. M., 2020, In : Hypertension (dallas, Tex. : 1979). 75, 2, p. 561-568 8 p. Methods for production of ergothioneine Borodina, I., Kell, D., Van Der Hoek, S. & Darbani Shirvanehdeh, B., 5 Nov 2020, IPC No. C12R 1/ 865 A N, Patent No. WO2020221795, 29 Apr 2020, Priority date 30 Apr 2019, Priority No. EP20190171749 Research output: Patent Prevalence of readily detected amyloid blood clots in 'unclotted' Type 2 Diabetes Mellitus and COVID-19 plasma: a preliminary report Pretorius, E., Venter, C., Laubscher, G. J., Lourens, P. J., Steenkamp, J. & Kell, D. B., 2020, In : Cardiovascular Diabetology. 19, 8 p., 193. Structural Similarities between Some Common Fluorophores Used in Biology, Marketed Drugs, Endogenous Metabolites, and Natural Products O'Hagan, S. & Kell, D. B., 2020, In : Marine Drugs. 18, 11, 18 p., 582. The biology of ergothioneine, an antioxidant nutraceutical Borodina, I., Kenny, L. C., McCarthy, C. M., Paramasivan, K., Pretorius, E., Roberts, T. J., van der Hoek, S. A. & Kell, D. B., 2020, In : Nutrition Research Reviews. 33, 2, p. 190-217 The Biology of Lactoferrin, an Iron-Binding Protein That Can Help Defend Against Viruses and Bacteria Kell, D. B., Heyden, E. L. & Pretorius, E., 2020, In : Frontiers in Immunology. 11, 1221. VAE-Sim: A novel molecular similarity measure based on a variational autoencoder Samanta, S., O'Hagan, S., Swainston, N., Roberts, T. J. & Kell, D. B., 2020, In : Molecules. 25, 15, 3446. Research output: Contribution to journal > Journal article > Research > peer-review Engineering the Yeast Saccharomyces cerevisiae for the Production of L-(+)-Ergothioneine van der Hoek, S. A., Darbani, B., Zugaj, K. E., Prabhala, B. K., Biron, M. B., Randelovic, M., Medina, J. B., Kell, D. B. & Borodina, I., 2019, In : Frontiers in Bioengineering and Biotechnology. 7, 262. Involvement of multiple influx and efflux transporters in the accumulation of cationic fluorescent dyes by Escherichia coli Jindal, S., Yang, L., Day, P. J. & Kell, D. B., 2019, In : BMC Microbiology. 19, 1, 16 p., 195. The role and robustness of the Gini coefficient as an unbiased tool for the selection of Gini genes for normalising expression profiling data Wright Muelas, M., Mughal, F., O'Hagan, S., Day, P. J. & Kell, D., 2019, In : Scientific Reports. 9, 1, 21 p., 17960. Energetic evolution of cellular Transportomes Darbani, B., Kell, D. B. & Borodina, I., 2018, In : B M C Genomics. 19, 11 p., 418.
Start Year 2018
 
Description Daye 
Organisation Daye
Country United Kingdom 
Sector Private 
PI Contribution Member of Medical Advisory Board. Approached to join due to extensive research in the area of the gut microbiome.
Collaborator Contribution The company is developing and optimising intuitive, scientifically robust sexual and reproductive health products.
Impact The company have created and marketed new products.
Start Year 2019
 
Description Stellenbosch 
Organisation University of Stellenbosch
Country South Africa 
Sector Academic/University 
PI Contribution Extraordinary (Honorary) Professor , Department of Physiological Science, Stellenbosch University Collaborative research in the areas of anti-microbial resistance, membrane transporters, dormant microbes as elements of supposedly non-communicable diseases, iron dysregulation, and enzyme improvement.
Collaborator Contribution Sharing of knowledge and analytical facilities at Stellenbosch, Manchester and Liverpool.
Impact Press release: Research opens door into new treatment options for chronic inflammatory diseases http://www.sun.ac.za/english/Lists/news/DispForm.aspx?ID=5104 Bezuidenhout J, Venter C, Roberts T, Tarr G, Kell D, Pretorius E (2020. Detection of citrullinated fibrin in plasma clots of RA patients and its relation to altered structural clot properties, disease-related inflammation and prothrombotic tendency. Front Immunol 11, 577523. DOI 10.3389/fimmu.2020.577523. bioRxiv link. DOI:10.1101/2020.05.28.121301. Venter C, Bezuidenhout JA, Laubscher GJ, Lourens PJ, Steenkamp J, Kell DB, Pretorius E (2020). Erythrocyte, platelet, serum ferritin and P-selectin pathophysiology implicated in severe hypercoagulation and vascular complications in COVID-19. Int J Mol Sci 21, 8234. DOI: 10.3390/ijms21218234 Pretorius E, Venter C, Laubscher GJ, Lourens PJ, Steenkamp J, Kell DB (2020) Prevalence of readily detected amyloid blood clots in 'unclotted' Type 2 Diabetes Mellitus and COVID-19 plasma: A preliminary report. Cardiovasc Diabetol 19, 193. DOI 10.1186/s12933-020-01165-7. Prevalence of amyloid blood clots in COVID-19 plasma. medRxiv 2020.2007.2028.20163543v20163541. DOI 10.1101/2020.07.28.20163543. Grobler C, Maphumulo SC, Grobbelaar LM, Bredenkamp J, Laubscher J, Lourens PJ, Steenkamp J, Kell DB, Pretorius E. (2020). COVID-19: The Rollercoaster of Fibrin(ogen), D-dimer, von Willebrand Factor, P-selectin and Their Interactions with Endothelial Cells, Platelets and Erythrocytes. Int J Mol Sci 21:5168. DOI: 10.3390/ijms21145168. Preprint at Grobler C, Bredenkamp J, Grobbelaar M, Maphumulo S, Laubscher J, Steenkamp J, Kell DB, Pretorius E (2020): COVID-19: The Rollercoaster of Fibrin(ogen), D-dimer, von Willebrand Factor, P-selectin and their interactions with endothelial cells, platelets and erythrocytes. Preprints.org 2020:202007.200142/v202001. Nunes JM, Fillis T, Page MJ, Venter C, Lancry O, Kell DB, Windberger U, Pretorius E (2020) Gingipain R1 and lipopolysaccharide from Porphyromonas gingivalis have major effects on blood clot morphology and mechanics. Front Immunol. 11, 1551. DOI: 10.3389/fimmu.2020.01551 Olsen I, Kell DB, Pretorius E (2020). Is Porphyromonas gingivalis involved in Parkinson's disease? Eur J Clin Microbiol Infect Dis, https://doi.org/10.1007/s10096-020-03944-2. DOI: 10.1007/s10096-020-03944-2. Kell DB, Heyden EL Pretorius, E. (2020) The biology of lactoferrin, an iron-binding protein that can help defend against viruses and bacteria. FrontiersIn Immunol 11, 1221. DOI: 10.3389/fimmu.2020.01221. Borodina I, Kenny LC, McCarthy CM, Paramasivan K, Pretorius R, Roberts TJ, van der Hoek SA, Kell DB (2020). The biology of ergothioneine, an antioxidant nutraceutical. Nutr Res Rev. 33, 190-217. Pubmed 32051057. DOI: 10.1017/S0954422419000301 de Villiers S, Bester J, Kell DB, Pretorius E (2019) Erythrocyte health and the possible role of amyloidogenic blood clotting in the evolving haemodynamics of female migraine-with-aura pathophysiology: Results from a pilot study. Frontiers Neurol 10, 1262. de Villiers S, Bester J, Kell DB, Pretorius E: Erythrocyte health and the possible role of amyloidogenic blood clotting in the evolving haemodynamics of female migraine-with-aura pathophysiology: Results from a pilot study. Cephalalgia 2019; 39:150-151 Adams B, Nunes JM, Page MJ, Roberts T, Carr J, Nell TA, Kell DB, Pretorius E (2019) Parkinson's disease: a systemic inflammatory disease accompanied by bacterial inflammagens. Front Aging Neurosci 11, 210. Preprint: bioRxiv 2019:646307v646301. DOI: 10.3389/fnagi.2019.00210 Visser MJE, Kell DB, Pretorius E (2019): Bacterial dysbiosis and translocation in psoriasis vulgaris. Front Cell Infect Microbiol 7, 9. DOI: doi: 10.3389/fcimb.2019.00007 Pretorius L, Kell DB, Pretorius E. (2018). Iron dysregulation and dormant microbes as causative agents for impaired blood rheology and pathological clotting in Alzheimer's type dementia. Frontiers in Neurosci 12, 851. DOI: 10.3389/fnins.2018.00851 de Waal GM, Engelbrecht L, Davis T, de Villiers WJS, Kell DB, Pretorius E (2018) Correlative Light-Electron Microscopy detects lipopolysaccharide and its association with fibrin fibres in Parkinson's Disease, Alzheimer's Disease and Type 2 Diabetes Mellitus. Sci. Rep. 8, 16798. DOI: 10.1038/s41598-018-35009-y. Pretorius E, Bester J, Page MJ, Kell DB (2018): The potential of LPS-binding protein to reverse amyloid formation in plasma fibrin of individuals with Alzheimer-type dementia. Frontiers Aging Neurosci 10, 257. DOI: 10.3389/fnagi.2018.00257 Kell DB, Pretorius E (2018) To what extent are the terminal stages of sepsis, septic shock, SIRS, and multiple organ dysfunction syndrome actually driven by a toxic prion/amyloid form of fibrin? Semin Thromb Hemost 44, 224-238. PMID 28778104. DOI: 10.1055/s-0037-1604108. PRIZE for MOST VIEWED Open Access paper in the journal that year Kell DB, Pretorius E (2018) No effects without causes. The Iron Dysregulation and Dormant Microbes hypothesis for chronic, inflammatory diseases: evidence and consequences. Biol Rev 93, 1518-1557. PMID 29575574. doi: 10.1111/brv.12407 Pretorius E, Page MJ, Mbotwe S, Kell DB (2018) Lipopolysaccharide-binding protein (LBP) can reverse the amyloid state of fibrin seen or induced in Parkinson's disease. PlosOne 13, e0192121. DOI: 10.1371/journal.pone.0192121 Pretorius E, Page MJ, Hendricks L, Nkosi NB, Benson SR, Kell DB (2018) Both lipopolysaccharide and lipoteichoic acids potently induce anomalous fibrin amyloid formation: assessment with novel Amytracker™ stains. J R Soc Interface 20170941. Pubmed. DOI: 10.1098/rsif.2017.0941 Pretorius, E., Page, M.J., Engelbrecht, L., Ellis, G.C., and Kell, D.B. (2017). Substantial fibrin amyloidogenesis in type 2 diabetes assessed using amyloid-selective fluorescent stains. Cardiovasc Diabetol 16, 141. PMID 29096623. doi: https://doi.org/10.1186/s12933-017-0624-5. Pretorius E, Mbotwe S, Kell DB (2017) Lipopolysaccharide-binding protein (LBP) reverses the amyloid state of fibrin seen in plasma of type 2 diabetics with cardiovascular comorbidities. Sci Rep 7, 9680 doi:10.1038/s41598-017-09860-4 Pretorius E, Page MJ, Henricks L, Nkosi NB, Benson SR & Kell DB (2017) Both lipopolysaccharide and lipoteichoic acids potently induce anomalous fibrin amyloid formation: assessment with novel Amytracker™ stains. BioRxiv 143867. DOI: https://doi.org/10.1101/143867 Kell, D. B. & Pretorius, E. (2017). Proteins behaving badly. Substoichiometric molecular control and amplification of the initiation and nature of amyloid fibril formation: lessons from and for blood clotting. Progr Biophys Mol Biol 123, 16-41. PMID 27554450. DOI: 10.1016/j.pbiomolbio.2016.08.006. Pretorius, E., Akeredolu, O.-O., Soma, P. & Kell, D.B. (2017). Major involvement of bacterial components in rheumatoid arthritis and its accompanying oxidative stress, systemic inflammation and hypercoagulability. Exp Biol Med. 242, 355-373. PMID 27889698. DOI: 10.1177/1535370216681549
Start Year 2017