Iruplinalkib (WX‑0593), a novel ALK/ROS1 inhibitor, overcomes crizotinib resistance in preclinical models for non-small cell lung cancer

Iruplinalkib displayed inhibitory effects in vitro in ALK-/ROS-positive cell lines

Iruplinalkib was designed to overcome resistance mutations identified in the clinic, especially ALK and ROS1. There was no chiral center in the iruplinalkib structure (Fig. 1a). In kinase assays, iruplinalkib inhibited the total tyrosine autophosphorylation of ALKWT, ALKL1196M, ALKC1156Y and EGFRL858R/T790M mutant with IC50 values in the range of 5.38–16.74 nM, and showed similar inhibitory potencies with brigatinib (Fig. 1b).

Fig. 1figure 1

Iruplinalkib inhibited kinase activity and the downstream signaling pathways of ALK and ROS1 in vitro. (a) Chemical formula of iruplinalkib. (b) Iruplinalkib and brigatinib inhibited the kinase activity of ALKWT, ALKL1196M, ALKC1156Y, and EGFRL858R/T790M. (c) The kinase selectivity of iruplinalkib and brigatinib to the fusion protein. Proliferation of Karpas299 (NPM-ALK), Ba/F3, Ba/F3 (EML4-ALKWT), Ba/F3 (EML4-ALKL1196M), Ba/F3 (EML4-ALKC1156Y), NCI-H1975 (EGFRL858R/T790M), HCC-78 (SLC34A2-ROS1), NIH-3T3 (CD74-ROS1), and Ba/F3 (SLC34A2-ROS1) cells. (d) The inhibitory effects of iruplinalkib on the phosphorylation of ALK, PI3K/AKT, RAS/MAPK and JAK/STAT3 pathways in NCI-H3122 cells, respectively. (e) Iruplinalkib inhibited phosphorylation of ROS1, PI3K/AKT and RAS/MAPK pathways in NIH-3T3 cells stably expressing CD74-ROS1, respectively. (f) Iruplinalkib inhibited phosphorylation of ROS1, RAS/MAPK and SHP-1 pathways in Ba/F3 cells stably expressing SLC34A2-ROS1, respectively

The selectivity of iruplinalkib against a number of non-ALK kinases was further evaluated. The results showed that iruplinalkib potently inhibited WT EGFR (IC50, 35 nM), but did not inhibit other 26 kinases strongly (Supplementary Table 3).

The activity of iruplinalkib on cell viability was next examined in several cell lines. In models harboring WT ALK fusion proteins [Karpas 299 (NPM-ALK) and Ba/F3 (EML4-ALKWT)], iruplinalkib exhibited similar cell growth inhibitory potency compared with brigatinib (IC50, 4–9 nM). However, in Ba/F3 cells expressing EML4-ALKL1196M or EML4-ALKC1156Y fusion proteins, iruplinalkib was slightly more potent. Cell growth IC50 values for iruplinalkib were 9.5 and 9 nM, compared with 19.5 and 14 nM, respectively, for brigatinib.

In Ba/F3 cells expressing WT SLC34A2-ROS1 fusion protein, iruplinalkib had comparable activity with brigatinib (IC50, 4.074 and 6.487 nM, respectively). However, HCC-78 cells expressing WT SLC34A2-ROS1 fusion protein, NCI-H1975 cells with EGFRL858R/T790M and NIH-3T3 cells with CD74-ROS1 expressed rendered resistance to iruplinalkib and brigatinib (IC50, 227.6–508 nM; Fig. 1c). Overall, iruplinalkib was a potent ALK/ROS1 inhibitor in cellular assays, and selectively inhibitory to engineered cell lines with ALK or ROS1 rearrangements.

Iruplinalkib inhibited the phosphorylation of downstream signaling molecules

Results showed that, after 2 h incubation with iruplinalkib, the phosphorylation levels of ALK, AKT, ERK but not STAT3 were reduced, and the raitos of phosphorylated protein to total protein were decreased in a dose-dependent manner, which was similar to brigatinib (Fig. 1d). It suggested that iruplinalkib inhibited the growth of NCI-H3122 cells by inhibiting the phosphorylation of ALK and RAS/MAPK/ERK, PI3K/AKT but not JAK/STAT3 pathways.

It is reported that ROS1-mediated cell signaling pathways include RAS-RAF-MEK-ERK, PI3K-AKT-mTOR and JAK-STAT3 pathways [35]. According to reports and the differences in the expression of signaling pathway proteins in different cells, the key signaling proteins in different signaling pathways were selected for research. Results showed that, after 2 h incubation with iruplinalkib, the phosphorylation levels of ROS1, AKT, ERK in NIH-3T3 (CD74-ROS1) cells and ROS1, ERK, SHP-1 in Ba/F3 (SLC34A2-ROS1) cells were inhibited, and the raitos of phosphorylated protein to total protein were dose-dependent decreased, which was similar to crizotinib (Fig. 1e). It suggested that iruplinalkib also inhibited of ROS1 and RAS/MEK/ERK, PI3K/AKT and SHP-1 pathways (Fig. 1f).

Iruplinalkib exhibits robust antitumor effects in xenograft models with ALK fusion and ROS1 fusion

Since ALK fusion is a important oncogenic driver fusion in NSCLC, we tested the antitumor effects of iruplinalkib and other TKIs on different xenograft models with different fusion types of ALK kinase. The LU-01-0015 (HIP-ALK) and NCI-H3122 xenograft tumor models were used, respectively. Iruplinalkib, crizotinib, and brigatinib had no effect on the body weight of different nude mouse xenograft models (all P ≥ 0.05, Fig. 2a-b). As we expected, iruplinalkib, brigatinib, crizotinib, and ceritinib were all able to inhibit tumor growth (all P < 0.05). Iruplinalkib exhibited a dose-dependent effect and was similar to brigatinib (at the same dose) in all two models, and all were superior to crizotinib (Fig. 2c-d; see Supplementary Tables 4–5 for details).

Fig. 2figure 2

Body weight and antitumor effect of iruplinalkib on BALB/c nude mouse xenograft model and the phosphorylation level of downstream signal molecules in vivo. (a) Body weight and relative body weight change of patient-derived tumor xenograft model LU-01-0015. (b) Body weight change of EML4-ALK transfected NCI-H3122 cells subcutaneously transplanted tumor mode; Antitumor effect of iruplinalkib on BALB/c nude mouse xenograft model. (c) LU-01-0015 (HIP1-ALK) tumor model; (d) EML4-ALK transfected NCI-H3122 cells subcutaneously transplanted tumor model. (e) The phosphorylation level of ALK, AKT, ERK, STAT3 and STAT5 in the tumor tissue in the NCI-H3122 model 8 h after the administration. NS (not significant): P ≥ 0.05. *: P < 0.05. **: P < 0.01. ***: P < 0.001

Iruplinalkib exerted antitumor effects by inhibiting ALK phosphorylation and JAK/STAT pathway in vivo

In order to check how iruplinalkib exerts its anti-tumor effects in vivo, tumor tissue samples were collected at 1 and 8 h after the last dose, and the phosphorylation level of downstream signaling pathways molecules were detected. Take NCI-H3122 xenograft model as example, results at 8 h after administration showed that iruplinalkib and brigatinib had similar inhibitory effects on the phosphorylation of ALK, AKT and ERK, which were similar to the in vitro data (Fig. 2e). Interestingly, unlike in the in vitro model, the levels of phosphorylation of STAT3 and STAT5 were found decreased after treating with both iruplinalkib and brigatinib (Fig. 2e). A smaller inhibition of phosphorylation was obersvered at 1 h after administration (Supplementary Fig. 1). The above data suggested that iruplinalkib may inhibit tumor growth by inhibiting ALK phosphorylation and JAK/STAT pathway in NCI-H3122 nude mouse xenograft model.

Plasma protein binding rate

At three test concentrations (0.2, 2 and 10 µM), the plasma protein binding rate of iruplinalkib is as follows (Fig. 3a). The recovery rates of iruplinalkib in the dialysis device were 81.8–99.9%, and the remaining rates in plasma after incubation for 5 h were 95–109.3%, indicating that the compound is stable in plasma during dialysis. Besides, iruplinalkib showed high protein binding rate at 0.2 µM in plasma of SD rats and showed medium protein binding rate at 0.2, 2 and 10 µM in plasma of CD-1 mice, SD rats, Beagle dogs, Macaca fascicularis and human. With the increase of concentration, the protein binding rates of iruplinalkib in rat plasma decreased gradually, showing a certain concentration dependence, and there was no concentration dependence of protein binding rate observed in other species plasma.

Fig. 3figure 3

(a) The plasma protein binding rate of iruplinalkib in CD-1 mice, SD rats, Beagles, Macaca fascicularis and human. (b) Induction of metabolic enzymes by iruplinalkib

Drug-drug interactions for iruplinalkib

Human hepatocytes derived from three donors were used for evaluating for hepatic enzyme inducing activity. The in vitro results showed that iruplinalkib was not an inducer of CYP1A2, CYP2B6, and CYP3A4 at 10 µM, and the cytochrome P450 isoenzyme CYP1A2 has not been induced. The gene expression related to cytochrome P450 isoenzyme CYP3A4 in hepatocytes of one of the three donors increased with iruplinalkib 1 µM. When concentration increased to 10 µM, it did not significantly induced CYP2B6 gene expression. In donor 1,  the folds of expression were 0.798, 1.36, and 2.62 times, respectively at 0.1, 1, and 10 µM; In donor 2, the folds were 0.862, 0.926, and 4.02 (> 4), respectively; In donor 3, those were 0.526, 1.13, and 0.602, respectively. The induction multiples of CYP3A4 gene expression in donor 1 were 0.621, 2.99, and 2.38 times, respectively at 0.1, 1 and 10 µM; In donor 2, those were 0.879, 1.80, and 2.81, respectively; In donor 3, those were 0.964, 9.36 (> 4), and 1.86, respectively (Fig. 3b).

Drug transporters for iruplinalkib

The results of interaction studies with SLC transporters suggested that iruplinalkib can inhibit the uptake activity of the transporters OATP1B1 and OATP1B3. Although iruplinalkib showed a high effective inhibition concentration, it also had obvious dose-dependent characteristics, no substrate activity was found (see Supplemental Tables 6–7). Iruplinalkib had some interference effect on the uptake of OAT1, but no dose-dependent relationship, so it can not be its inhibitor (Supplemental Tables 8–9). Unlike the transporter MATE1, iruplinalkib can effectively inhibits its uptake activity, and this effect was dose-dependent (Supplemental Table 10). Using a stably transfected cell line of HEK293 expressing transporter OATP1B1, OATP1B3 or OCT2, formal substrate investigation tests were performed for three concentration gradients (30 µM, 3 µM, 0.3 µM) of iruplinalkib. The results indicated that the uptake transport activity ratios of the three relevant transporters for this compound were all less than two, and showed non-dose dependent characteristics (Supplemental Table 11). The test results indicated that iruplinalkib was not an uptake substrate for OATP1B1, OATP1B3, OCT2, OAT1 and OAT3.

The evaluation of the inhibitory effect of iruplinalkib on the uptake of transporter OATP1B1 and OATP1B3 showed that this inhibitory effect was real, despite its relatively high effective inhibitory concentration. It should be noted that even at low concentrations, iruplinalkib still has a certain inhibitory or interference effect when atorvastatin was used as a substrate (Fig. 4a-b). The inhibitory effect of iruplinalkib on transporter OCT2 uptake below 10 µM was an interference rather than an inhibitory effect (Fig. 4c). And with metformin as the substrate, this case cannot be fitted with a suitable inhibition curve, indicating that iruplinalkib had only a weak inhibitory effect on OCT. The results also showed that iruplinalkib was a stronger inhibitor of transporter MATE1 and MATE2K (Fig. 4d-e).

Fig. 4figure 4

(a-e) Inhibition of OATP1B1-293, OATP1B3-293, OCT2-293, MATE1-293, and MATE2K-293 uptake activity by iruplinalkib; (f) Inhibition of BCRP efflux activity by iruplinalkib on Caco-2 cells

In vitro studies revealed that iruplinalkib is a transported substrate of P-glycoprotein (P-gp or ABCB1) not for breast cancerresistance protein (BCRP). The ABC efflux transporter inhibitor test was carried out with Caco-2 cell line as the model, which showed that iruplinalkib was a BCRP inhibitor (Table 1). Using 10 µM E3S as the substrate, iruplinalkib significantly inhibited its efflux, and the overall efflux ratio gradually decreased with increasing concentration, showing a dose-dependent characteristic. The results of the fitted inhibition curves are shown (Fig. 4f). Digoxin at 5 µM was used as a substrate, and it showed mostly non-dose-dependent and somewhat disruptive effects on P-gp efflux below 3 µM; at the concentration points of 10 µM and 30 µM, it showed effective with 89.8% and 98.7%, respectively (Table 1). The experimental results showed that iruplinalkib was not an efflux inhibitor of BSEP and MRP2, nor did it have a dose-dependent profile (supplementary Tables 12–13).

Table 1 The results of iruplinalkib as P-gp and BCRP substrate, BCRP and P-gp inhibitor investigation

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