More than two million severe adverse drug reactions are estimated to occur in the United States each year, a quarter of which are attributable to interactions between co-administered drugs.18–20
An increasing number of studies report DDIs resulting from the inhibition of membrane transporters, adding to the complexity of drug safety assessment. To date, however, we lack the arsenal of selective probes necessary to mechanistically interpret transporter-mediated DDIs. Further, to our knowledge there have been no large screens of registered drugs to prospectively predict transporter based DDIs.
Here, we sequentially assessed inhibition of the renal organic cation transporter OCT2, counter-screened for inhibition of other major transporters, and prioritized hits based on their clinical plasma concentrations. Eighty-nine of the 244 identified OCT2 inhibitors are among the top 200 most prescribed drugs in the United States (IMS Health, accessed at http://www.rxlist.com
). For several FDA-approved drugs OCT2 inhibition would occur near the therapeutically active plasma concentrations, meriting caution when co-administered with drugs whose renal excretion relies on OCT2 transport. Recently, the International Transporter Consortium proposed criteria that would trigger a clinical DDI study based on in vitro interaction studies with membrane transporters.21
Because of enormous interspecies differences in the tissue distribution and substrate and inhibitor specificity of transporters, the International Transporter Consortium recommended that clinical studies, rather than animal studies be conducted. Six drugs in our screen met these criteria for inhibition of OCT2, including cimetidine, which is the most extensively studied inhibitor of renal organic cation transport and the perpetrator in many of the reported clinical renal DDIs.5, 22–25
For inhibitors of OCT2, metformin was recommended as a model substrate in a clinical study. Clearly, based on our studies, such studies are warranted.
Using computational analyses, we showed that inhibitors of OCT2 are found across multiple structural and pharmacological classes and that inhibitor potency is not associated with substructures that define series of structurally related compounds, but rather to broader molecular properties like positive charge, lipophilicity, molecular size and flexibility. This is similar to the structure-inhibition relationship for the hepatic organic cation transporter OCT1, for which lipophilicity, positive atomic charge and hydrogen bond donors were predictive of inhibitors.26
Compounds from three distinct clusters that differ in molecular properties resulted in OCT2 inhibition, suggesting that the clusters may represent complementary inhibitory mechanisms. For example, known transported substrates of OCT2 are almost exclusively found in cluster II (; Supporting Information, Table S3
), suggesting competitive binding to the transport binding site of cluster II inhibitors; further, corticosterone, which is prototypical of the structures in our cluster III, results in an allosteric modulation of substrate binding to OCT2 and OCT1;27
and inhibitors in cluster I are distinct from the others in their larger size and flexibility, implying that inhibition may be caused by occlusion of the substrate binding site as suggested in crystallographic experiments for LeuT, a bacterial homologue of human SLC6 transporters.28–29
The pronounced importance of positive charge for the tentatively substrate-like inhibitors in cluster II is in line with the demonstrated tendency for substantial renal secretion of charged drugs.1
In contrast, lipophilicity is not a hallmark of actively secreted drugs;1, 30
its importance for the inhibitory endpoint studied here is however not surprising, given the well-established influence of hydrophobic interactions between ligands and proteins.
Notably, only 7 of the 52 compounds that inhibited OCT2 near the total clinical plasma concentration also inhibited its hepatic paralog OCT1. Because the two transporters are highly homologous especially in regions where circulating inhibitors are likely to bind, our data imply that the overall homology between OCT2 and OCT1 does not directly reflect homology in the binding site. For example, a single-residue switch in rabbit OCT2, from the OCT2 specific glutamate to the glutamine of OCT1, altered cimetidine binding to an OCT1-like phenotype.31
Our results also suggest that the affinity for OCT1 is consistently lower than for OCT2. A previous report of OCT1 inhibitors among registered drugs confirms this phenomenon (Supporting Information, Figure S2
: the majority of the compounds that specifically inhibited OCT2 at 20µM also inhibit OCT1 at the higher concentration of 100µM. We suggest that the hepatic OCT1 transporter, which experiences high portal vein concentrations of orally absorbed compounds, through evolutionary mechanisms is less susceptible to circulating inhibitors.
Six drugs were identified that inhibit OCT2 at clinical concentrations of free drug, with inhibition potencies ranging from 0.6 to 23 µM (; ). Of interest is that 5 of the potential in vivo probe inhibitors are eliminated exclusively (>95%) through metabolism (Supporting Information
, Table S1
). Previously, inhibitors of renal excretion of basic drugs were thought to be eliminated largely by the kidney. Our data suggest that drugs that are extensively metabolized may also be important inhibitors of renal secretion of basic drugs. Interestingly, we confirmed that cimetidine is a more potent inhibitor of the apically located MATE transporters than of OCT2 (; ).32– 33
Our in vitro data for MATE1 and MATE2-K, in addition to those for OCT2 (), suggest the necessity of conducting a clinical trial based on the proposed guidelines.21
Potency of putative clinical inhibitors in HEK293 cells expressing reference OCT2, the genetic variant OCT2-A270S, OCT1, MATE1 or MATE2-K.
OCT2 governs the entry of many circulating toxins into the tubular epithelium, including the neurotoxin 1-methyl-4-phenylpyridinium (MPP+
), the DNA intercalator ethidium and the herbicide paraquat,8, 34–35
with the cellular uptake typically coupled to efflux into the urine via MATEs.34
Selective inhibition of OCT2 or MATEs would thus result in opposing effects on cellular toxin accumulation, decreasing and exacerbating renal toxicity, respectively. Accordingly, protection from cisplatin-induced nephrotoxicity was recently demonstrated in OCT2-deficient mice, and the reduced-function genetic variant OCT2-A270S was associated with decreased acute nephrotoxicity in cisplatin-treated patients.36
The selective inhibitors identified here may thus hold therapeutic potential as cytoprotectants in anticancer drug therapy. It is noteworthy that the co-administration of an inhibitor of organic anion transport, probenecid, to reduce the renal toxicity of the antiviral agent cidofovir is recommended in the official product label.37–38
In summary, we present the largest available resource of organic cation transporter inhibition data, identifying 244 OCT2 inhibitors among prescription drugs with six potential drugs that can be used as in vivo probes of renal transport or in protection against drug-induced nephrotoxicity.