Selpercatinib (LOXO292) and pralsetinib (BLU667) are RET protein tyrosine kinase inhibitors (TKIs) recently approved for treating RET-altered cancers. However, RET mutations that confer selpercatinib/pralsetinib resistance have been identified, necessitating development of next-generation RET TKIs. While acquired RET G810C/R/S/V mutations were reported in selpercatinib-treated patients, it was unclear whether all of these and other potential G810 mutants are resistant to selpercatinib and pralsetinib. Here, we profiled selpercatinib and pralsetinib on all six possible G810 mutants derived from single nucleotide substitution and developed novel alkynyl nicotinamide-based RET TKIs to inhibit selpercatinib/pralsetinib-resistant RET G810 mutants. Surprisingly, the G810V mutant found in a clinical study was not resistant to selpercatinib or pralsetinib. Besides G810C/R/S, G810D also conferred selpercatinib/pralsetinib resistance. Alkynyl nicotinamide compounds such as HSN608, HSL476, and HSL468 have better drug-like properties than alkynyl benzamides. Six of these compounds inhibited all six G810 solvent-front mutants and the V804M gatekeeper mutant with IC50 < 50 nmol/L in cell culture. Oral administration of HSN608 at a well-tolerated dose (30 mg/kg) gave plasma level > 30x the IC50s of inhibiting all G810 mutants in cell culture. In cell-derived xenograft tumors driven by KIF5B-RET (G810C) that contains the most frequently observed solvent-front mutant in selpercatinib-treated patients, HSN608, HSL476, and HSL468 significantly suppressed and caused regression of the selpercatinib-resistant tumors. This study clarifies the sensitivities of different RET solvent-front mutants to selpercatinib and pralsetinib and identifies novel alkylnyl nicotinamide-based RET TKIs for inhibiting selpercatinib/pralsetinib-resistant G810 mutants.

This article is featured in Highlights of This Issue, p. 691

The rearranged-during-transfection (RET) gene encodes a protein tyrosine kinase (PTK). Oncogenic mutations and gene fusions result in constitutively activated RET kinase. RET alterations are most often found in thyroid cancers and in non–small cell lung cancers (NSCLC; refs. 1, 2), but also increasingly found in diverse cancer types, such as pancreatic, colorectal, and breast cancers (3–7). RET-altered cancers respond poorly to immunotherapy (8). Earlier clinical studies of RET-targeted therapy used protein tyrosine kinase inhibitors (TKI) of low RET inhibitor potency, which might not adequately suppress the RET kinase activity in the tumors (1, 9, 10). These TKIs also suffer from resistance caused by the RET gatekeeper V804 mutants. More recently, clinical studies showed that the potent RET TKIs selpercatinib and pralsetinib gave high response rates in RET-altered thyroid cancers and NSCLC, and the responses were considered durable (11–14). Consequently, selpercatinib and pralsetinib were approved by the FDA as the first two RET-targeted therapy drugs in late 2020. Furthermore, based on the basket trial of selpercatinib in RET fusion-positive tumors (15), selpercatinib was approved by FDA recently as a tumor-agnostic drug for pan-RET fusion-positive solid tumors.

In addition to target bypass mechanisms (16–19), secondary target mutations have repeatedly been found as a mechanism of acquired resistance to PTK-targeted cancer therapy (20–22). A strategy to overcome the on-target mechanism of resistance is to develop new generations of TKIs capable of inhibiting these drug-adapted mutations (22–24). Although selpercatinib and pralsetinib are effective on the RET gatekeeper mutants, preclinical experiments by us showed that G810C/S/R located at the solvent-front site of the RET kinase domain gave the strongest selpercatinib and pralsetinib resistance (25). Moreover, G810C/S/R/V mutations have been reported in patients who developed resistance to selpercatinib following the initial response (18, 25, 26), and the G810C/S mutants were reported in patients with NSCLC who acquired resistance to pralsetinib (27).

While we have shown experimentally that G810C/S/R mutations confer resistance to selpercatinib and pralsetinib, it is unclear if G810V and other potential G810 mutations are resistant to selpercatinib or pralsetinib. We reasoned that single nucleotide substitutions of the RET G810 codon GGC are more likely to occur than double and triple nucleotide substitutions. Therefore, we focused our analysis of G810 mutation spectrum on single nucleotide substitutions. Besides G810C/S/R/V, G810A, and G810D missense mutations could also arise by single nucleotide substitution of the G810 codon. G810A was identified previously by us as a vandetanib-resistant mutant (28). In this study, we profiled sensitivities of selpercatinib and pralsetinib on the panel of these six RET G810 mutations to identify and clarify the spectrum of G810 mutants on selpercatinib and pralsetinib resistance.

Previously, we reported that a small library of TKIs containing alkynyl nicotinamide scaffold, which is more drug-like than the corresponding first-generation alkynyl benzamides, potently inhibited RET kinase (29). This prior study was completed before the issue of RET solvent-front mutations were known, so the compounds were not evaluated against these mutations. Motivated by the success of the initial alkynyl nicotinamides, HSN608 and analogs, to inhibit CCDC6-RET potently (sub-nanomolar IC50 values), we proceeded to synthesize additional analogs, including HSL468 and 476, and screened the library of alkynyl nicotinamide compounds by a cell-based assay of KIF5B-RET kinase-dependent cells containing V804M, G810C, G810R, and G810S mutants. Seven compounds were selected for IC50 profiling of the six RET G810 mutants and the V804M gatekeeper mutant. These compounds showed potent inhibition of all G810 mutants and the V804M mutant. Pharmacokinetic (PK) study showed that HSN608 and other alkynyl nicotinamide-based RET TKIs are orally bioavailable and could reach plasma concentrations above those required to inhibit all G810 mutants and the V804M mutants. G810C was the most frequently observed selpercatinib-resistant RET mutant in patients with cancer (18, 25, 26). Four alkynyl nicotinamide-based RET TKIs were tested in RET (G810C) mutant-dependent cell-derived xenograft (CDX) tumors. Three of these compounds suppressed the selpercatinib-resistant tumors without apparent toxicity.

Reagents and chemicals

Antibodies to phospho-RET (Y905) (catalog no. 3221), cleaved-PARP (catalog no. 9541), and Flag (catalog no. 2368) were purchased from Cell Signaling Technology (Danvers, MA). Selpercatinib and pralsetinib were from Chemieteck (Indianapolis, IN). The purity of selpercatinib was > 99.5% determined by high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), and quantitative elemental analysis. The purity of pralsetinib was > 99% determined by achiral and chiral HPLCs, NMR, and quantitative elemental analysis. Ponatinib was from LC Laboratories (Woburn, MA). The purity was > 99% determined by HPLC and quantitative elemental analysis.

Alkynyl nicotinamide-based RET inhibitors

The detailed chemical synthesis information and analytic data are provided in Supplementary information. Generally, unless otherwise stated, reagents and anhydrous solvents were purchased from widely available commercial sources and used as received without further purification. The 1H and 13C NMR spectras (Supplementary Fig. S1) were recorded in DMSO-d6 or Methanol-d4 as solvents using a 500 MHz spectrometer. Me4Si was used as an internal standard. Chemical shifts reported in parts per million (δ) downfield. 1H NMR spectral data reported as follows: chemical shift (δ ppm) [multiplicity, coupling constant (Hz), integration]. Multiplicities are reported as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, or combinations thereof. High-resolution mass spectra (HRMS) were analyzed using electron spray ionization technique and as time-of-flight (TOF) mass analyzer. All the new synthesized compounds were characterized using 1H NMR, 13C NMR, and HRMS data. The purity of all tested compounds was ≥ 95% and analyzed by an Agilent HPLC using C18 column 5C18-MS-II COSMOSIL (4.6ID × 250 mm; Supplementary Table S1).

KIF5B-RET mutation constructs, cell lines, and cell-culture assays

The BaF3 mouse pre-B cell line was originally obtained from Dr. H.G. Wang in 2000 and stored in liquid nitrogen tanks. The identity of the BaF3 cells was evaluated on the basis of their dependence on mouse interleukin-3 for cell survival (28). BaF3 cells expressing KIF5B-RET, KIF5B-RET G810A, G810C, G810R, G810S, and V804M were as reported (9, 17, 25, 28, 30, 31). KIF5B-RET G810D and G810V mutations were generated by PCR-based mutagenesis with primers containing the desired mutations. Mutations were confirmed by DNA sequencing. BaF3 cells expressing these new KIF5B-RET mutants were established using a lentiviral vector similar to that described (9). Cells were routinely checked for free of Mycoplasma using the Universal Mycoplasma Detection Kit (ATCC, #30–1012K).

The cell viability assay was performed using CellTiter-Glo reagent (Promega, Madison, WI) in 96-well plates as described previously (9, 25). Drug screening and IC50 data of B/KR and mutation cells were from two or more experiments performed in triplicates (n > 6). For immunoblotting analysis of pRET, cells were treated with the drug for 4 hours at concentrations indicated in the figure legends. For cleaved PARP analysis, cells were treated with the drug for 16 hours. Cell lysate preparation and analysis by immunoblotting were performed as described (9, 25).

In vitro kinase assay

In vitro kinase assays were performed by a contract service (Reaction Biology, Malvern, PA) using purified recombinant RET kinase domain proteins produced by baculoviruses in insect cells as GST fusion proteins. The reaction mixture contained 20 mmol/L Hepes, pH 7.5, 10 mmol/L MaCl2, 1 mmol/L EGTA, 0.02% Brij35, 0.02 mg/mL BSA, 0.1 mmol/L Na3VO4, 2 mmol/L DTT, 1% DMSO, 10 μmol/L 32P-ATP, 20 μmol/L CHKtide (KKKVSRSGLYRSPSMPENLNRPR), plus indicated recombinant RET proteins. Phosphorylated peptide was detected by filter-binding method. The IC50 assays were performed with a 10-dose IC50 assay with a 3-fold serial dilution starting at 1 μmol/L. The in vitro kinase assays were performed in duplicate. Kinase selectivity profiling was performed by Eurofins Contract Research Organization.

Absorption, distribution, metabolism, and excretion and PK studies

These experiments were performed by a contract research organization (Pharmaron, Beijing, China).

Animal studies

Animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Oklahoma Health Sciences Center. B/KR or B/KR(G810C) cells were tested free of 20 mouse pathogens (IDEXX, Columbia, MO) and Mycoplasma. Cells (1 × 107 cells/0.1 mL/each) were inoculated subcutaneous into the right flank of approximately 5-week-old female SHO mice (Charles River, Wilmington, MA). After tumor formation, mice in the same cage were randomly assigned to different groups (6 mice/group in 2 cages), and treated with vehicle, pralsetinib, selpercatinib, or test compounds at the indicated doses by oral gavage once a day. Tumor size and animal body weight were measured as described with a caliper and a scale (30). Tumor sizes at the endpoint were compared between different groups, and between the same group at the start of treatment.

Data and statistical analysis

IC50 curve fitting was performed using GraphPad Prism 9 by the equation Y = Ymax/(1+(X/IC50)Hillslope as described (32), where X: concentration; Y: relative remaining activity; Ymax: activity of mock-treated control group. Statistical analysis of tumor sizes was performed using unpaired Mann–Whitney test. A difference with P < 0.05 was considered statistically significant.

Data availability

The data that support the findings of this study are available in the paper and the supplementary materials. The raw data are available from the corresponding authors upon reasonable request.

Ponatinib is an alkynyl benzamide, which contains imidazo[1,2-b]pyridazine hinge binder and has been shown to inhibit RET kinase in vitro. However, ponatinib is a potent inhibitor of the human ether-a-go-go-related gene (hERG) protein (IC50 0.8–2 μmol/L), highly protein bound (greater than 99%), poorly soluble in water (insoluble at pH 6.8; Table 1). hERG IC50 is an indicator of cardiac toxicity. A hERG IC50 < 1 μmol/L is considered to have a high potency, whereas an IC50 > 10 μmol/L is considered to have a low potency. Compounds that typically bind hERG contain basic amines that can be protonated, such as the piperazine found in ponatinib, and hydrophobic moieties (33). In our prior work, we had determined that deleting the piperazine moiety in ponatinib to a non-basic residue abrogated RET inhibition (29). We speculated the conversion of the hydrophobic benzamide found in ponatinib to the hydrophilic nicotinamide moiety might reduce affinity for hERG without affecting RET inhibition. To arrive at compounds that are different from ponatinib, we also changed the hinge binding moiety in ponatinib, imidazo[1,2-b]pyridazine, into other hinge binders, such as isoquinoline, naphthyridines, pyrimidine, pyridine, pyrido[3,4-b]pyrazine and 1H-pyrazolo[3,4-b]pyridine (Fig. 1A). Kinase active sites contain chiral amino acid residues and so it is plausible to achieve targeting using compounds with stereogenic centers. Thus, we also included two compounds, HSN670 and HSN671, which are enantiomers.

Table 1.

Ion channel inhibition, protein binding activity, and solubility of test compounds.

IC50 (μmol/L)Solubility in PBS (μmol/L)
CompoundhERG (K+)Nav1.5 (Na+)Cav1.2 (L-type Ca2+)Protein binding (Human/mouse or rat)pH1.2pH4.5pH6.8
HSN608 >30 >30 >30 99%/99% 289.4 107.9 0.07 
HSL476 2.4 nd nd 94%/96% 103.7 294.3 315.9 
HSL468 23.7 nd nd 94%/92% 304.7 294.0 117.0 
HSND17 nd nd nd nd 290.8 276.6 11.1 
Ponatiniba 0.8–2 nd nd >99% nd nd nd 
Diclofenac nd nd nd nd 3.2 25.7 302.2 
IC50 (μmol/L)Solubility in PBS (μmol/L)
CompoundhERG (K+)Nav1.5 (Na+)Cav1.2 (L-type Ca2+)Protein binding (Human/mouse or rat)pH1.2pH4.5pH6.8
HSN608 >30 >30 >30 99%/99% 289.4 107.9 0.07 
HSL476 2.4 nd nd 94%/96% 103.7 294.3 315.9 
HSL468 23.7 nd nd 94%/92% 304.7 294.0 117.0 
HSND17 nd nd nd nd 290.8 276.6 11.1 
Ponatiniba 0.8–2 nd nd >99% nd nd nd 
Diclofenac nd nd nd nd 3.2 25.7 302.2 

Abbreviation: nd, not determined.

aFDA drug evaluation report, and ACS Med. Chem. Lett. (2020) 11, 491–496.

Figure 1.

Design and screen of alkynyl nicotinamide-based RET kinase inhibitors. A, Alkynyl nicotinamide compound library. Numbers in bracket (red) are calculated LogP (cLogP, ChemDraw version 20). B, General scheme for making compounds via Sonogashira reaction. C, Cell-based screening of indicated alkynyl nicotinamide compounds (20 nmol/L) in B/KR, B/KR(V804M), B/KR(G810C), B/KR(G810R), and B/KR(G810S) cells. D, Comparison of HSN670 and HSN671 on B/KR cells. E, IC50 of HSN608 and HSL476 on RET (G810C), RET(G810R), and RET(G810S) enzyme activities determined by an in vitro kinase assay.

Figure 1.

Design and screen of alkynyl nicotinamide-based RET kinase inhibitors. A, Alkynyl nicotinamide compound library. Numbers in bracket (red) are calculated LogP (cLogP, ChemDraw version 20). B, General scheme for making compounds via Sonogashira reaction. C, Cell-based screening of indicated alkynyl nicotinamide compounds (20 nmol/L) in B/KR, B/KR(V804M), B/KR(G810C), B/KR(G810R), and B/KR(G810S) cells. D, Comparison of HSN670 and HSN671 on B/KR cells. E, IC50 of HSN608 and HSL476 on RET (G810C), RET(G810R), and RET(G810S) enzyme activities determined by an in vitro kinase assay.

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The compounds were synthesized via Sonogashira reaction, following our published protocol (Fig. 1B; 29). The calculated logP values (cLogP) of the analogs bearing nicotinamide and different hinge binders were lower than the cLogP of ponatinib (Fig. 1A), hinting that these analogs could be better drug-like than ponatinib. Indeed, this was borne out experimentally. For example, the HSN608 IC50 for hERG and other ion channels [such as Nav1.5 (Na+) and Cav1.2 L-type (Ca2+)] was > 30 μmol/L, which is among the safest drugs, while the hERG IC50s of HSL476 and HSL468 were 2.4 and 23.7 μmol/L (Table 1). Some of these compounds also have low protein binding and high aqueous solubility (Table 1).

With a library of novel alkynyl nicotinamides in hand, we proceeded to evaluate the inhibition of mutant RET by the compounds. RET G810C/S/R mutants BaF3/KIF5B-RET (B/KR) cells depend on the RET kinase activity for survival and proliferation (28), thus we used these cell lines to evaluate the efficacy of the synthesized compounds against mutant RET. We previously established B/KR cells containing the solvent-front G810C, G810R, and G810S mutants and the V804M gatekeeper mutant (9, 25). Using these RET kinase-dependent cell lines, we screened the alkynyl nicotinamide compound library (Fig. 1A) at 20 nmol/L and included ponatinib, selpercatinib, and pralsetinib as controls (Fig. 1C). Selpercatinib and pralsetinib did not inhibit these three solvent-front mutants, consistent with their resistance to selpercatinib and pralsetinib (25, 26). While ponatinib gave > 50% inhibition of G810 (KR wild-type) and G810S, it had < 25% inhibition on V804M, G810C, and G810R. HSND17 and HSL476 inhibited all four mutants > 50%, while several other compounds, such as HSN721, HSN608, and HSL468, had > 50% inhibition of all but G810R cells (Fig. 1C). Interestingly, the type of chirality (R or S) at the pyrrolidine moiety (HSN670 and HSN671) did not affect RET inhibition as both compounds had similar inhibition profile (Fig. 1D). Seven alkynyl nicotinamide compounds (HSND17, HSN576, 608, 632, 721, HSL468 and 476) were chosen, based on bearing distinct hinge binding moieties (i.e., different chemotypes) as well as showing potent inhibition of RET mutants in B/KR mutant cells (Fig. 1C), for further analysis on a panel of six G810 mutants (see below) and the V804M gatekeeper mutant in the B/KR cells and B/KR cells containing these mutants (Table 2).

Table 2.

Sensitivity of test compounds on RET V804M and G810 mutants determined in B/KR and mutant cell lines.

IC50 of RET mutants (nmol/L)
CompoundWTV804MG810AG810CG810DG810RG810SG810V
HSND17 1.70 ± 0.08 14.08 ± 0.54 1.56 ± 0.07 11.37 ± 0.32 5.21 ± 0.36 24.53 ± 0.79 7.76 ± 0.33 4.10 ± 0.30 
HSN576 4.25 ± 0.25 20.43 ± 0.77 2.85 ± 0.10 17.76 ± 0.51 6.40 ± 0.34 46.47 ± 1.89 15.05 ± 0.50 7.30 ± 0.22 
HSN608 3.89 ± 0.13 18.96 ± 0.73 3.98 ± 0.30 19.04 ± 0.62 3.55 ± 0.24 28.84 ± 1.02 7.38 ± 0.13 8.60 ± 0.60 
HSN632 2.86 ± 0.12 34.89 ± 1.19 3.57 ± 0.25 32.12 ± 1.41 6.71 ± 0.30 14.05 ± 0.44 10.63 ± 0.35 4.79± 0.26 
HSN721 2.84 ± 0.18 11.22 ± 0.63 3.57 ± 0.11 14.95 ± 0.44 2.22 ± 0.11 82.72 ± 2.12 11.44 ± 0.31 8.17 ± 0.44 
HSL468 3.49 ± 0.16 20.30 ± 1.09 3.89 ± 0.07 12.24 ± 0.63 1.47 ± 0.05 46.75 ± 1.11 8.02 ± 0.21 8.34 ± 0.36 
HSL476 1.76 ± 0.04 15.05 ± 0.48 1.88 ± 0.06 11.56 ± 0.30 7.98 ± 0.27 17.99 ± 0.37 10.07 ± 0.38 3.60 ± 0.15 
Ponatinib 13.75 ± 1.19 111.5 ± 5.47 8.84 ± 0.21 50.33 ± 4.53 10.10 ± 0.64 124.9 ± 9.39 33.94 ± 2.21 24.10 ± 9.45 
Selpercatinib 14.96 ± 0.66 127.40 ± 6.42 180.10 ± 14.6 1371 ± 153.6 254.10 ± 11.5 2580 ± 97.26 940.30 ± 37.34 25.71 ± 0.76 
Pralsetinib 19.46 ± 1.00 23.75 ± 1.02 85.13 ± 3.26 956.50 ± 60.8 499.90 ± 17.0 7939 ± 214.9 565.9 ± 17.71 53.19 ± 1.83 
IC50 of RET mutants (nmol/L)
CompoundWTV804MG810AG810CG810DG810RG810SG810V
HSND17 1.70 ± 0.08 14.08 ± 0.54 1.56 ± 0.07 11.37 ± 0.32 5.21 ± 0.36 24.53 ± 0.79 7.76 ± 0.33 4.10 ± 0.30 
HSN576 4.25 ± 0.25 20.43 ± 0.77 2.85 ± 0.10 17.76 ± 0.51 6.40 ± 0.34 46.47 ± 1.89 15.05 ± 0.50 7.30 ± 0.22 
HSN608 3.89 ± 0.13 18.96 ± 0.73 3.98 ± 0.30 19.04 ± 0.62 3.55 ± 0.24 28.84 ± 1.02 7.38 ± 0.13 8.60 ± 0.60 
HSN632 2.86 ± 0.12 34.89 ± 1.19 3.57 ± 0.25 32.12 ± 1.41 6.71 ± 0.30 14.05 ± 0.44 10.63 ± 0.35 4.79± 0.26 
HSN721 2.84 ± 0.18 11.22 ± 0.63 3.57 ± 0.11 14.95 ± 0.44 2.22 ± 0.11 82.72 ± 2.12 11.44 ± 0.31 8.17 ± 0.44 
HSL468 3.49 ± 0.16 20.30 ± 1.09 3.89 ± 0.07 12.24 ± 0.63 1.47 ± 0.05 46.75 ± 1.11 8.02 ± 0.21 8.34 ± 0.36 
HSL476 1.76 ± 0.04 15.05 ± 0.48 1.88 ± 0.06 11.56 ± 0.30 7.98 ± 0.27 17.99 ± 0.37 10.07 ± 0.38 3.60 ± 0.15 
Ponatinib 13.75 ± 1.19 111.5 ± 5.47 8.84 ± 0.21 50.33 ± 4.53 10.10 ± 0.64 124.9 ± 9.39 33.94 ± 2.21 24.10 ± 9.45 
Selpercatinib 14.96 ± 0.66 127.40 ± 6.42 180.10 ± 14.6 1371 ± 153.6 254.10 ± 11.5 2580 ± 97.26 940.30 ± 37.34 25.71 ± 0.76 
Pralsetinib 19.46 ± 1.00 23.75 ± 1.02 85.13 ± 3.26 956.50 ± 60.8 499.90 ± 17.0 7939 ± 214.9 565.9 ± 17.71 53.19 ± 1.83 

In addition to the G810C/R/S mutants, G810A, G810D, and G810V mutants are three other G810 mutants that can arise by a single nucleotide substitution of the human RET G810 codon (GGC). G810V was reported as an acquired mutation in a patient with NSCLC who developed selpercatinib resistance, but its contribution to selpercatinib or pralsetinib resistance was not determined experimentally (26). G810A was isolated previously as a vandetanib-resistant mutant (28). Therefore, we generated new B/KR(G810V) and B/KR(G180D) cell lines and examined sensitivity of all six G810 mutants to selpercatinib and pralsetinib. Surprisingly, G810V was not resistant to either selpercatinib or pralsetinib (Table 2, Fig. 2A; Supplementary Fig. S2). G810A was not resistant to pralsetinib and was weakly resistant to selpercatinib with a 15-fold higher IC50. In contrast, G810D caused modest 21-fold resistance to selpercatinib and 30-fold resistance to pralsetinib in our cell-based assay (Table 2, Fig. 2A; Supplementary Fig. S2).

Figure 2.

Sensitivities of different G810 mutants derived from single nucleotide substitutions to RET TKIs. A, IC50 curves of B/KR (G810 wild-type) and G810 mutant cells to pralsetinib, HSN608, and HSL476. B, Immunoblotting analyses of RET TKIs on inhibition of KR and its G810 mutant kinase activity and induction of apoptosis in B/KR and G810 mutant cells. For examination of pRET, cells were treated with the indicated concentrations of the RET TKIs for 4 hours, whereas for cPARP analysis, cells were treated for 16 hours. PST, pralsetinib.

Figure 2.

Sensitivities of different G810 mutants derived from single nucleotide substitutions to RET TKIs. A, IC50 curves of B/KR (G810 wild-type) and G810 mutant cells to pralsetinib, HSN608, and HSL476. B, Immunoblotting analyses of RET TKIs on inhibition of KR and its G810 mutant kinase activity and induction of apoptosis in B/KR and G810 mutant cells. For examination of pRET, cells were treated with the indicated concentrations of the RET TKIs for 4 hours, whereas for cPARP analysis, cells were treated for 16 hours. PST, pralsetinib.

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The seven alkynyl nicotinamide compounds had lower nanomolar IC50s in B/KR cells that were > 2-time more potent than ponatinib, selpercatinib, and pralsetinib (Table 2). Except HSN721, which had an IC50 > 50 nmol/L in G810R cells that was 28-fold higher than that in the G810 wild-type cells, these alkynyl nicotinamide compounds were able to inhibit all six G810 mutant cells with IC50 < 50 nmol/L (Table 2, Fig. 2; Supplementary Fig. S2), which is several folds lower than the IC50 for the approved RET drugs for G810R (selpercatinib: 2,580 nmol/L, pralsetinib: 7,939 nmol/L). Moreover, all seven alkynyl nicotinamide compounds inhibited the ponatinib-resistant V804M mutant cells (ponatinib IC50: 96 nmol/L) with IC50s < 35 nmol/L. Two compounds (HSN608 and HSL476) were tested in in vitro kinase assay on RET G810C/S/R. Consistent with cell-based sensitivity assessment, both compounds displayed low nanomolar IC50s on these three G810C/S/R solvent-front mutants (Fig. 1E).

Immunoblotting assay were performed on selected compounds and cell lines (Fig. 2B; Supplementary Fig. S2B) using phospho-RET (Y905) as a biomarker of RET tyrosine kinase activity and cleaved PARP as a biomarker of apoptosis. As shown in Fig. 2B, pralsetinib inhibited V804M, G810A, and G810V and caused apoptosis of these cells, whereas it did not inhibit G810C, G810R, G810D, G810S at the tested concentrations and did not induce apoptosis of these cells. Similar results were obtained with selpercatinib (Supplementary Fig. S2). In comparison, HSN608 and HSL476 were able to inhibit G810C, G810R, G810D, and G810S and induced apoptosis of these cells (Fig. 2B), indicating that these compounds could inhibit these pralsetinib- and selpercatinib-resistant G810 mutants.

Kinome panel experiments (obtained at the Contract Research Organization, Eurofins, Supplementary Table S2 and S3, Supplementary Fig. S3) show that the compounds have similar kinome profile to the approved drug, ponatinib, with some interesting differences: (i) our compounds bind to RET kinase better than ponatinib; and (ii) ponatinib binds to these additional kinases, CDK8, EPHA3, EPHA8, HUNK, LTK, MAP4K2 better than the nicotinamide compounds.

Absorption, distribution, metabolism, and excretion-PK analyses of HSN608 showed that, in liver microsomes [(protein) = 0.5 mg/mL], HSN608 had T1/2 = 2.2, 1.4, 3.2 hours for rats, dogs and humans, respectively (Supplementary Table S4), translating into CLint = 5.4, 8.3, 3.6 μL/min/mg, respectively. HSL476 and HSL468 had shorter T1/2 in liver microsomes with corresponding higher CLint (Supplementary Table S3). A PK study in rats showed that HSN608 was orally bioavailable (F ∼30%; Fig. 3A), with a reasonable T1/2 of 3.4 hours (2 mg/kg, intravenously). At 30 mg/kg by oral gavage, the Cmax was 460 ng/mL (843 nmol/L; Fig. 3A), which was 30–45x of the HSN608 IC50 for inhibition of RET V804M and G810C/R mutants, and 70–240x of the HSN608 IC50 for other RET G810 mutants in the B/KR mutant cells (Fig. 3A, Table 2). A second PK study in rats was performed on HSL468, which had a F of 49% at 10 mg/kg in oral gavage (Fig. 3B), T1/2 of 4.3 hours, and a Cmax 199 ng/kg that was similar to that of HSN608 at the same oral dose.

Figure 3.

PK studies of HSN608 and HSL468 in rats. PK study in rats. Mean plasma concentrations over time profile for HSN608 (A) after 2 mg/kg i.v., 10 mg/kg p.o. and 30 mg/kg p.o., or for HSL468 (B) after 2 mg/kg i.v. and 10 mg/kg p.o. Tables under the graphs are the estimated PK parameters.

Figure 3.

PK studies of HSN608 and HSL468 in rats. PK study in rats. Mean plasma concentrations over time profile for HSN608 (A) after 2 mg/kg i.v., 10 mg/kg p.o. and 30 mg/kg p.o., or for HSL468 (B) after 2 mg/kg i.v. and 10 mg/kg p.o. Tables under the graphs are the estimated PK parameters.

Close modal

RET G810C was the most frequently detected acquired RET mutants in selpercatinib-treated patients with cancer whose tumors progressed after a period of response, and therefore was chosen for our tumor experiments in animals. To determine if alkynyl nicotinamide class of RET TKIs are effective on the selpercatinib/pralsetinib-resistant RET G810C tumors, we tested four compounds in the B/KR(G810C) CDX tumors similar to that described recently (30). B/KR (non-mutant) and B/KR(G810C) cells formed rapidly growing tumors approximately 2 weeks after subcutaneous inoculation (Fig. 4A). In the control experiment, selpercatinib and pralsetinib administered (25 mg/kg, every day) by oral gavage caused B/KR tumor regression (Fig. 4A and B, left). However, the selpercatinib treatment was ineffective on B/KR(G810C) tumors, whereas HSN608, HSL476, and HSND17 (25 mg/kg, every day, oral gavage) significantly (P < 0.001) inhibited the B/KR(G810C) tumors in the same experiment (Fig. 4A and B, middle). In another experiment, B/KR(G810C) CDX tumors were treated by oral gavage with HSL468 (25 mg/kg, every day) or HSN608 (50 mg/kg, every day). While the vehicle-treated tumors grew rapidly, HSL468 and HSN608 caused tumor regression (Fig. 4A and B, right). Animal body weight monitoring showed that treatment with HSND17, but not with other compounds, had a 10% reduction in the body weight (Supplementary Fig. S4). Thus, all but HSND17 were tolerated well by the animals.

Figure 4.

In vivo activity of alkynyl nicotinamide-based RET TKIs on B/KR(G810C) CDX tumors. Mice bearing B/KR(G810C) CDX tumors (1 tumor/mouse, 5 or 6 mice/group) were treated with the test compounds at the indicated doses by daily oral gavage. A, Tumor dimensions were measured with a caliper, and tumor sizes were estimated using the formula: tumor size = (length × width × width)/2. The data shown are the mean ± SD. B, Waterfall plots of tumor size changes at the endpoint from the baseline on the start date of drug treatment. Each bar corresponds to a tumor. ns, not statistically different; **, P < 0.01. PST, pralsetinib; SPC, selpercatinib. C, Immunoblot of tumor tissue lysates with the indicated antibodies. D, Images of tumors collected at the endpoint of experiment 3 (presented in the right panels of A, B, and C). E, Hematoxylin- and eosin-stained tumor tissue section.

Figure 4.

In vivo activity of alkynyl nicotinamide-based RET TKIs on B/KR(G810C) CDX tumors. Mice bearing B/KR(G810C) CDX tumors (1 tumor/mouse, 5 or 6 mice/group) were treated with the test compounds at the indicated doses by daily oral gavage. A, Tumor dimensions were measured with a caliper, and tumor sizes were estimated using the formula: tumor size = (length × width × width)/2. The data shown are the mean ± SD. B, Waterfall plots of tumor size changes at the endpoint from the baseline on the start date of drug treatment. Each bar corresponds to a tumor. ns, not statistically different; **, P < 0.01. PST, pralsetinib; SPC, selpercatinib. C, Immunoblot of tumor tissue lysates with the indicated antibodies. D, Images of tumors collected at the endpoint of experiment 3 (presented in the right panels of A, B, and C). E, Hematoxylin- and eosin-stained tumor tissue section.

Close modal

Analysis of tumor tissues by immunoblotting showed that pRET and the downstream pErk1/2 (Fig. 4C, left) was inhibited by selpercatinib and pralsetinib in the B/KR tumor samples, indicating that the KIF5B-RET kinase activity in the B/KR CDX tumors were inhibited by these drugs. However, selpercatinib could not inhibit the KR(G810C) kinase activity in the tumors (Fig. 4C, middle), whereas HSN608, HSND17, and HSL476 suppressed pRET in the B/KR(G810C) tumors. Little RET protein was detected in the immunoblot from samples of the tiny tumor tissues obtained at the endpoint from HSL468-treated mice (Fig. 4C right, 4D middle), indicating that these tumor samples were composed of contaminated host tissue. This also explains why pErk1/2 in these tissues was not reduced. In support of this notion, contaminated host muscle was observed by histologic examination of a small tumor (Fig. 4D and E).

Selpercatinib and pralsetinib were recently developed RET TKIs that gave durable response in RET-altered thyroid cancer, NSCLC, and other solid tumors (11–15). Limited clinical data published so far indicated that the G810 residue located at the solvent-front of the RET kinase domain is the predominant site of on-target mutations that cause selpercatinib and pralsetinib resistance (18, 25–27). However, the spectrum of G810 mutations that may cause selpercatinib- and/or pralsetinib-resistant was unclear. In this study, we profiled all six possible G810 mutations resulted from single nucleotide substitution of the human RET G810 codon. Our data showed that G810V detected previously in the plasma cell-free tumor DNA (ctDNA) of a KIF5B-RET+ patient with NSCLC who developed selpercatinib resistance (26) was not resistant to selpercatinib or pralsetinib. This result suggests that G810C, which was the dominant mutation in the early stage of developing resistance, and G810R that appeared later were responsible for the selpercatinib-resistant in this patient. G810C was also the predominant RET mutation in a patient with medullary thyroid cancer who developed resistance to selpercatinib and the only RET mutation in a CCDC6-RET+ NSCLC who developed selpercatinib resistance (25). In addition to G810C/R/S, we show here that G810D is also resistant to selpercatinib and pralsetinib. These results clarify the spectrum of RET G810 mutations resistant to selpercatinib and pralsetinib, which should help to guide the development of the new RET TKIs.

A lesson learned from other PTK-targeted therapies is that the next generations of drugs can be used to prolong the duration of disease control when the mechanism of resistance was drug-adapted target mutations (22–24). In fact, a macrocyclic TKI TPX-0046 (34), which is effective on RET G810C/S/R mutant, is in phase I/II clinical trials (NCT04161391; ref. 35). However, the RET gatekeeper V804M mutant is resistant to TPX-0046 (34, 36).

Ponatinib is an FDA-approved drug, which inhibits RET but suffers from many limitations including poor solubility, and a high affinity for hERG. In addition, ponatinib is a poor inhibitor of RET gatekeeper mutants and G810C/R solvent-front mutations. In medicinal chemistry, nitrogen scanning is a technique used to improve both the physicochemical properties of drugs as well as enhancement of affinity for target enzymes (37). In this study, we synthesized a series of alkynyl nicotinamide compounds (“necessary nitrogen” analog of the benzamide found in ponatinib) bearing different hinge binders and different substitution patterns. Inhibition of RET G810 mutants was sensitive to various substitutions. For example, the naphthyridine class displayed differential inhibition based on the ring nitrogen position. While HSN608 (1,7-naphthyridin-8-amine) and HSN721 (2,7-naphthyridin-1-amine), potently inhibited RET V804M, G810C and G810S, other naphthyridines such as HSN722 (2,6-naphthyridin-1-amine) and HSN756 (1,6-naphthyridin-5-amine) were poor inhibitors of RET V804M, G810C, and G810S. In general methyl substituted nicotinamides were less potent than the analogs without methyl substitution. For example, HSN804, HSN742, HSN576, and HSN700 (non-methyl substituted nicotinamides) were better inhibitors of mutant RET than the corresponding methyl substituted analogs, HSN805, HSN806, HSN431, and HSN580 respectively. For compounds containing the pyrido[3,4-b]pyrazine moiety, dimethyl substituted compounds (HSND16, HSL211, and HSL476) were more potent than diethyl substituted compounds (HSL507 and HSND15). Methyl groups are considered as “magic” in medicinal chemistry as there are several instances whereby the addition of a methyl group to compounds improves activity (38). For compounds containing monocyclic hinge binders, pyrimidine (HSL468) was better than pyridine-containing HSL815, HSN816, and HSN631. From our screening effort, we identified three alkynyl nicotinamide scaffold-based RET TKIs (HSN608, HSL476, and HSL468) that are potent inhibitors of RET G810 mutants and display potent activity on the V804M mutant. These alkynyl nicotinamide compounds, particularly HSN608 and HSL468, have high hERG IC50s that are in the range of the safest drugs. They displayed reasonably good liver microsome stability and PK property as drug candidates. These compounds are orally bioavailable, and were able to inhibit the selpercatinib-resistant RET (G810C) CDX tumors. Thus, our study has identified candidates of novel RET TKIs for overcoming the selpercatinib/pralsetinib-resistant RET G810 mutants and for the potential TPX-0046-resistant V804M mutant.

F.W. Holtsberg reports being a shareholder in KinaRx, Inc. M.J. Aman reports other support from KinaRx, Inc. during the conduct of the study and other support from KinaRx, Inc. outside the submitted work. H.O. Sintim reports grants from the NIH during the conduct of the study; other support from KinaRx, Inc. outside the submitted work; in addition, H.O. Sintim has a patent for US11001559B2 issued and licensed to KinaRx, Inc. J. Wu reports grants from the NIH, Oklahoma Center for the Advancement of Science and Technology, Oklahoma Tobacco Settlement Endowment Trust, and Stephenson Endowment; and grants from the NIH during the conduct of the study; in addition, J. Wu has a patent for a provisional patent pending. No disclosures were reported by the other authors.

U. Khatri: Data curation, formal analysis, validation, investigation, writing–original draft, writing–review and editing. N. Dayal: Data curation, validation, investigation, writing–original draft, writing–review and editing. X. Hu: Data curation, formal analysis, investigation, writing–original draft, writing–review and editing. E. Larocque: Data curation, investigation, writing–review and editing. N. Naganna: Data curation, investigation, writing–review and editing. T. Shen: Data curation, investigation, writing–review and editing. X. Liu: Data curation, investigation, writing–review and editing. F.W. Holtsberg: Funding acquisition, project administration, writing–review and editing. M.J. Aman: Funding acquisition, project administration, writing–review and editing. H.O. Sintim: Conceptualization, resources, data curation, formal analysis, supervision, funding acquisition, validation, investigation, visualization, methodology, writing–original draft, project administration, writing–review and editing. J. Wu: Conceptualization, resources, data curation, formal analysis, supervision, funding acquisition, validation, investigation, visualization, methodology, writing–original draft, project administration, writing–review and editing.

This work was supported by NIH grants R01CA273168 and R41CA250707, and in part by NIH grant R01CA242845 and Oklahoma Center for the Advancement of Science and Technology grant HR19–026, the Oklahoma Tobacco Settlement Endowment Trust, and the Peggy and Charles Stephenson Endowed Chair fund. The shared resources at the University of Oklahoma Health Sciences Center were supported by NIH grants P20GM103639 and P30CA225520. We also acknowledge support from the Purdue University Center for Cancer Research (PCCR), NIH P30CA023168, PCCR Pilot Grant award, and Purdue Institute for Drug Discovery.

The publication costs of this article were defrayed in part by the payment of publication fees. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).

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Supplementary data