Adapted with permission from Ref. 123.
A machine learning‐based chemoinformatic analysis of the altered metabolites in each knockout revealed distinct differences in substrate specificity [39]. For example, compared to Oat1, Oat3 appears to prefer larger, more hydrophobic (endogenous) molecules with a larger number of rings. This raises the possibility that one can exploit this molecular/structural data inherent to the substrates to gain insight into Oat‐mediated transport. This approach, which represents a strategy for designing inhibitors or for designing drugs targeting specific Oats, analyzes the substrates that interact with the Oats and identifies common chemical features/molecular determinants among the substrates to generate hypothetical models called pharmacophore models. Pharmacophore models for Oat1, Oat3, and Oat6 have been built, and these have proven quite helpful in increasing our understanding of transporter–substrate interactions [36, 120,130–133]. For example, initial computational characterization and comparative molecular field analyses of the substrate binding preferences of Oat1 versus Oat6 suggested that the (mostly) pharmaceutical substrate selectivity of these transporters was influenced by mass, net charge, and hydrophobicity [131]. Other approaches have based pharmacophores on endogenous metabolites, which accumulate in the plasma of Oat1 knockout animals [120, 132]. In these studies, metabolites were clustered into distinct groups and consensus pharmacophores were created. Chemical databases were screened virtually for molecules which fit the pharmacophores, and several of these molecules were subsequently shown to interact with Oat1 in in vitro assays [120, 132]. In addition, to analyze the cationic binding potential of Oat3, a pharmacophore model based on cations that bind Oat3 was created. The model was used to screen a commercially available compound database, leading to the identification of molecules that proved to be potent highly selective in vitro inhibitors of Oat3 [36].
4.4.3 Inhibitors
Substrates have been more difficult to determine for OATs because of the demand for radiolabeled substrates or mass spectrometry‐based analysis methods. A popular alternative to these types of studies is to perform inhibition assays, where potential interacting molecules are screened for inhibition of classic OAT function, often with probe substrates or fluorescent substrates. Due to the multi‐specific nature of the OATs, several inhibitors of OAT function have been reported, yet specific inhibitors of the individual transporters have yet to be identified. Nevertheless probenecid, which is nonspecific, has historically been used as an inhibitor of OAT‐mediated anionic drug uptake and handling [23]. Probenecid does not appear to be transported by the OATs and is believed to block transport of other organic anions through its binding to the transporter which (in the case of the kidney) decreases renal excretion and enhances plasma retention of drugs, an example being the common practice of co‐administering probenecid to prolong the action of beta‐lactam antibiotics [23].
4.4.4 Natural Products and Herbal Medicines
While OAT‐inhibition research has mainly focused on drugs, metabolites, and toxicants, there has recently been interest in the role of OATs in handling traditional Chinese medicines, flavonoids, and natural products [134–136].From a structural perspective, these compounds differ from the traditional OAT substrates, as they are typically larger and more complex. Nonetheless, multiple in vitro studies using cells over‐expressing OATs have been published. OAT1 and OAT3 have similar profiles with respect to natural products and have both been shown to interact with ginkgolic acid, rosmarinic acid, apigenin, gincompounds such as 18β‐glycyrrhetinic acid, apigenin, wogonin, luteolin, epigallocatechin‐3‐gallate, and several others [137–141].There are some metabolites that appear to be more specific toward a certain transporter, however, as Oat3 is uniquely associated with epicatechin gallate [142] and OAT4 is uniquely associated with catechin [137]. Considering that many of these compounds are important in traditional Chinese medicine or present in common dietary items, it is important to understand that their interactions with OATs.
4.4.5 Drug–Drug Interactions and Drug–Metabolite Interactions
When two or more organic anion substrates are available for transport, it is believed that they compete and consequently perturb the transport of each other [23]. In fact, since many OAT substrates are endogenous metabolites or commonly prescribed pharmaceuticals, competition between substrates for transport is believed to represent an important source of drug–drug interaction (DDI) [88] or drug–metabolite interaction (DMI). For example, clearance of methotrexate has been reported to be reduced with NSAIDs acting as competitive substrates for the binding sites of renal OATs leading to an increase in plasma concentration of methotrexate and the manifestation of severe toxicity in the form of bone marrow suppression and damage to intestinal epithelium [4, 23]. Interestingly, it was found that individuals genetically predisposed to hyperuricemia had an increased risk of developing gout when taking diuretics compared with individuals without the predisposition [143]. Since the drugs and the metabolite associated with hyperuricemia, urate, are primarily handled by the OATs, it is possible that this increased risk is due to drug–metabolite interaction/competition at the level of the transporters. It is also worth noting that diet‐derived compounds, like the aforementioned natural products, may also compete with drugs and metabolites.
In addition to the DDI or DMI induced by the direct interaction of OAT substrates and inhibitors on the transporter itself, certain drugs, phytomedicines or xenobiotics, can alter OAT‐mediated disposition of clinically relevant substances through an indirect manner by modulating OAT expression and function. Many OAT substrates, including indoxyl sulfate and other indole‐based compounds, have been shown to activate the aryl hydrocarbon receptor (AHR), which may play a role in the expression of OATs [144, 145]. Recent studies showed that anticancer drugs ixazomib, oprozomib, delanzomib, bortezomib, and carfilzomib enhanced OAT expression and OAT‐mediated drug transport by inhibiting proteasomal activity, thereby preventing OAT degradation in this organelle [146].
4.5 SYSTEMS BIOLOGY OF OATS
4.5.1 Physiological Role
The main physiological role of the OATs