Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.822867
Title: A fragment based binary weapon approach to cellular drug targeting
Author: Grixti, Justine
ISNI:       0000 0005 0288 9752
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2017
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Abstract:
Drugs enter cells via proteinaceous membrane transporters which are normally used for the transportation of nutrients and metabolites. Drug efficacy is thereby mainly determined by the distribution and kinetics of influx and efflux transporters for which they are substrates. The idea that the transporter-mediated disposition of drugs can be altered by the addition of a second small molecule, that of itself has no inhibitory pharmacological effect, but that influences the expression of transporters for the primary drug, was studied. This strategy is here referred to as the 'binary weapon' approach. The experimental system tested the ability of such a molecule to increase the toxicity of the nucleoside analogue gemcitabine to pancreatic cancer cells. An initial phenotypic screen of a 500-member polar drug (fragment) library, using the MTT assay to estimate cell viability, yielded three candidate fragments which showed efficacy in enhancing the toxicity of gemcitabine by at least 10%. Cheminformatics analyses were used to screen the rest of the 2000 members of this library suite, and the structures of 20 fragments had a Tanimoto similarity (with MACCS encoding) >0.7 to the reference fragments. Wet-lab screening experiments were performed, and indeed each fragment was itself active (the cheminformatics thus providing for a massive enrichment). The top six representatives from the whole library, showing activity at the lowest concentration studied (3 uM), were considered to be the final active fragments. These fell into three clusters whose members bore reasonable structural similarities to each other (two were in fact isomers), lending strength to the self-consistency of both the predicted and experimental strategies used. Moreover, none of the six active fragments seemed to have any such effects on the non-cancerous pancreatic cell line hPDE, the human embryonic kidney cell line HEK293, or the neuroblastoma cell line SH-SY5Y, implying that there is, or can be, at least some degree of specificity in the 'binary weapon' approach. Given the experimental evidence that some of the Maybridge fragments were increasing gemcitabine's toxicity towards Panc1 cells, it was intriguing to understand the underlying mechanism of action. ENT1-3 influx and ABCC2, 3, 4, 5 and 10 efflux transporters displayed measurable transcripts in Panc1 cells, along with a ribonucleoside reductase RRM1 known to affect gemcitabine toxicity. Very strikingly, the addition of gemcitabine alone increased the expression of the transcript for ABCC2 by more than 12-fold, and that of RRM1 by more than fourfold, and each of the fragment 'hits' served to reverse this. However, MK571, an inhibitor of ABCC2, was without significant effect, possibly implying that the effect of RRM1 is the more significant contributor to the phenotypic changes noted. It proved difficult to find inhibitors of RRM1. It seems, therefore, that while the effects measured here were mediated more by efflux than influx transporters, and potentially by other means, the binary weapon idea is hereby fully confirmed: it is indeed possible to find molecules that manipulate the expression of transporters involved in the bioactivity of a pharmaceutical drug. This opens an entirely new area of chemical genomics-based, transport-mediated drug targeting. Moreover, internal experimental controls to ensure that unwanted off-target effects were kept to a minimum showed that many of the library fragments were indeed stimulating cell growth. To investigate this observation, phenotypic screening of the Maybridge fragment library, again using the MTT assay to estimate cell viability, and cheminformatics analyses to determine the Tanimoto similarity between fragments, yielded five molecules which enhanced cell growth by at least 10%, displaying potential uses in the biotechnology industry where the growth rate stimulation of mammalian cells is usually sought after. The efficacy of these fragments was further tested in the presence of gemcitabine. Only one of these fragments proved to be bimodal, enhancing Panc1 cell growth rate in the absence of gemcitabine by at least 10%, however, also showing efficacy in enhancing the drug's toxicity by the same percentage when treatment was administered in the presence of gemcitabine.
Supervisor: Kell, Douglas ; Day, Philip Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.822867  DOI: Not available
Keywords: phenotypic screening ; binary weapon ; cheminformatics ; gemcitabine ; anticancer drugs ; pancreatic cancer ; drug transporters
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