Enhancing Drug Efficacy and Therapeutic Index through Cheminformatics-Based Selection of Small Molecule Binary Weapons That Improve Transporter-Mediated Targeting: A Cytotoxicity System Based on Gemcitabine

The transport of drug molecules is mainly determined by the distribution of influx and efflux transporters for which they are substrates. To enable tissue targeting, we sought to develop the idea that we might affect the transporter-mediated disposition of small-molecule drugs via the addition of a second small molecule that of itself had no inhibitory pharmacological effect but that influenced the expression of transporters for the primary drug. We refer to this as a “binary weapon” strategy. The experimental system tested the ability of a molecule that on its own had no cytotoxic effect to increase the toxicity of the nucleoside analog gemcitabine to Panc1 pancreatic cancer cells. An initial phenotypic screen of a 500-member polar drug (fragment) library yielded three “hits.” The structures of 20 of the other 2,000 members of this library suite had a Tanimoto similarity greater than 0.7 to those of the initial hits, and each was itself a hit (the cheminformatics thus providing for a massive enrichment). We chose the top six representatives for further study. They 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 our conceptual and experimental strategies. Existing literature had suggested that indole-3-carbinol might play a similar role to that of our fragments, but in our hands it was without effect; nor was it structurally similar to any of our hits. As there was no evidence that the fragments could affect toxicity directly, we looked for effects on transporter transcript levels. In our hands, only the ENT1-3 uptake and ABCC2,3,4,5, and 10 efflux transporters displayed measurable transcripts in Panc1 cultures, 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 (MRP2) by more than 12-fold, and that of RRM1 by more than fourfold, and each of the fragment “hits” served to reverse this. However, an inhibitor of ABCC2 was without significant effect, implying that RRM1 was possibly the more significant player. These effects were somewhat selective for Panc cells. It seems, therefore, that while the effects we measured were here 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 that are involved in the bioactivity of a pharmaceutical drug. This opens up an entirely new area, that of chemical genomics-based drug targeting.