(A) Urinary excretion profiles for prescription drugs. Data on the urinary recovery of 308 drugs was collected from published clinical mass balance studies. Light blue bars denote the renal recovery of unchanged parent drug. For approximately one-half of all analyzed compounds, renal excretion of the parent compound contributes significantly to the overall elimination (light blue bars, ≥10% urinary excretion of parent drug). Data on the total recovery of parent drug and metabolites (dark blue bars) show that renal elimination is an important pathway also for drugs that are metabolically converted, with more than 90% of all analyzed drugs being recovered in urine to a significant extent (dark blue bars, ≥10% urinary excretion of parent drug + metabolites). (B) Schematic illustration of key drug transporters expressed in renal proximal tubule epithelium. Cation transporters are on the left hand side and anion transporters on the right. The basolateral membrane facing the blood is nearest to the reader. The nephron is shown in the far left.

(A) Schematic of the workflow applied to identify OCT2 inhibitors. The number of compounds analyzed in each step is noted on the arrows. [Inhibitor]: total plasma concentration of inhibitor. [Unbound inhibitor]: concentration of inhibitor not bound to plasma proteins. (B) Uptake of ASP⁺ in HEK293 cells stably expressing OCT2. (Bi) Time course of ASP⁺ (5 µM) uptake without (open circles) or with 500 µM cimetidine (open triangles), and in cells transfected with an empty vector (closed circles). The Z' factor at the sampling time used for screening is indicated in the figure. (Bii) Concentration dependence of ASP⁺ uptake in OCT2 expressing cells (open circles) and empty vector transfected cells (open triangles). The OCT2 specific uptake (closed circles) was calculated by subtracting the non-specific uptake in empty vector transfected cells from that in the cells expressing OCT2. (Biii) Inhibitory effects of cimetidine on ASP⁺ (5 µM) uptake in OCT2 expressing cells (open bars) and in cells transfected with an empty vector (closed bars). Data are presented as mean ± s.d. (three separate samples from one representative experiment).

(A) Overview of the results from the screening of OCT2 inhibition. Each bar represents one compound. 244 compounds resulting in at least 50% decreased uptake of ASP+ were classified as inhibitors (shaded in light gray). Data are presented as mean ± s.d. (samples in triplicate from one experiment). (B) Therapeutic classes of the screened compounds. Therapeutic classes with ≥ 10 members in the screening library are shown as individual bars; all other classes were combined (“other”). Shaded and white bars represent the number of OCT2 inhibitors and non-inhibitors in each class, respectively. (C) High-potency OCT2 inhibitors resulting in ≥ 95% inhibition at 20 µM, corresponding to estimated IC₅₀ ≤ 1µM.

(A) Correlation analyses between OCT2 inhibition and inhibition of OCT2-A270S, the hepatic homologue OCT1, and the apical organic cation transporters MATE1 and MATE2-K. The prototypical organic cation transport inhibitor cimetidine is indicated by the arrows. (B) Venn diagram showing the overlapping inhibitors for OCT2, OCT1, MATE1 and MATE2-K. (C) Selectivity of inhibition for putative clinical inhibitors of OCT2. The concentration dependent inhibition of ASP⁺ uptake is shown for HEK293 cells stably expressing OCT2-Reference (closed circles), OCT2-A270S (open circles), MATE1 (upward pointing triangles), MATE2-K (downward pointing triangles) and OCT1 (open squares). Data are presented as mean ± s.d. (three separate samples from one representative experiment).

(A) Distribution of key physicochemical properties of OCT2 inhibitors (yellow) and non-inhibitors (blue). Differences were assessed with the two-sided unpaired Student’s t-test assuming unequal variances and are presented as uncorrected p-values. (B) Substructure analysis of OCT2 inhibitors. The inset shows substructure fragments enriched in inhibitors compared to the entire dataset, with absolute and relative frequencies of each fragment in inhibitors and non-inhibitors. Enrichment was assessed with the hypergeometric test and is presented as uncorrected p-values. Significantly enriched fragments are highlighted in example OCT2 inhibitors. (C) Molecular descriptors discriminating between inhibitors and non-inhibitors. Bars show the mean PLS regression coefficients from 500 cross-validated models (100 random cross-validation partitionings with five segments each); standard errors are shown in light blue. Descriptors with positive coefficients have higher values in inhibitors, and descriptors with negative coefficients have higher values in non-inhibitors. The descriptors shown were included in at least 50% of the final 500 models after optimization through iterative exclusion of uninformative descriptors; inset thermometer plots show in black the fraction of all models that include the descriptor. (D) Internal and external prediction results. The left-hand plot shows the fraction of true positive and true negative predictions (averaged over all 500 models) from the inner cross-validation loop, during which model parameters and descriptor selections are optimized. The right-hand plot shows predictions from the outer cross-validation loop (averaged over 100 random partitionings of the dataset). The latter thus represent unbiased external predictions.

(A) Self-Organizing Map (SOM) clustering. Each hexagon is a cluster of similar molecules, and hexagons near each other are more similar than distant clusters. (Ai) Hexagons are colored according to the number of compounds they contain. (Aii) Hexagons are colored according to the similarity to their neighbors. Lighter color indicates greater similarity. (Aiii) Hexagons were merged into larger clusters if they were not significantly different (F-test p-value < 0.1). The hierarchical tree shows the chemical similarity relationships among the final clusters. (Aiv) Hexagons are colored according to the fraction of the compounds that inhibit at least 50% (left-hand SOM) or 75% (right-hand SOM) of OCT2-mediated transport. (Av) Enrichment of OCT2 inhibitors. Cluster colors are scaled to the negative log₁₀ of the uncorrected hypergeometric test p-values. Clusters with enrichment p-values > 0.05 are colored grey. (B) Molecular descriptors specifically important for inhibitors in clusters I, II and III. The descriptors shown are the nine with the most significantly different PLS regression coefficients among separate models of OCT2 inhibitors in clusters I, II or III, respectively. Bars show mean PLS regression coefficients for cluster I (purple), II (red) and III (blue); standard errors are shown in light blue. (C) Structural differences between molecules in clusters I, II and III. Thermometer plots show molecular properties of prototypical members of each cluster, with descriptors in the same order as in (B).