Pseudokinases are structurally similar to kinases but lack catalytic activity; instead, pseudokinases typically function as scaffolds, often promoting the degradation of substrate proteins by bringing them into close proximity with ubiquitin ligases. Two studies explored the structures and protein interactions of the pseudokinases TRIB1 (Jamieson et al.) and TRIB2 (Foulkes et al.). Their findings reveal new insights into the structural regulation of TRIB proteins and show that these proteins, which are implicated in leukemia and other cancers, can bind to clinically approved kinase inhibitors. Binding by these drugs caused structural changes in the TRIB proteins that deprotected them from degradation upon interacting with ubiquitin ligases, indicating that these drugs might be repurposed or redesigned to perturb the function of TRIBs in cancer patients.

A major challenge associated with biochemical and cellular analysis of pseudokinases is a lack of target-validated small-molecule compounds with which to probe function. Tribbles 2 (TRIB2) is a cancer-associated pseudokinase with a diverse interactome, including the canonical AKT signaling module. There is substantial evidence that human TRIB2 promotes survival and drug resistance in solid tumors and blood cancers and therefore is of interest as a therapeutic target. The unusual TRIB2 pseudokinase domain contains a unique cysteine-rich C-helix and interacts with a conserved peptide motif in its own carboxyl-terminal tail, which also supports its interaction with E3 ubiquitin ligases. We found that TRIB2 is a target of previously described small-molecule protein kinase inhibitors, which were originally designed to inhibit the canonical kinase domains of epidermal growth factor receptor tyrosine kinase family members. Using a thermal shift assay, we discovered TRIB2-binding compounds within the Published Kinase Inhibitor Set (PKIS) and used a drug repurposing approach to classify compounds that either stabilized or destabilized TRIB2 in vitro. TRIB2 destabilizing agents, including the covalent drug afatinib, led to rapid TRIB2 degradation in human AML cancer cells, eliciting tractable effects on signaling and survival. Our data reveal new drug leads for the development of TRIB2-degrading compounds, which will also be invaluable for unraveling the cellular mechanisms of TRIB2-based signaling. Our study highlights that small molecule–induced protein down-regulation through drug “off-targets” might be relevant for other inhibitors that serendipitously target pseudokinases.

Here, we found that the low-affinity ATP-binding site in TRIB2 ( 40 ) was druggable with small molecules previously described as ATP-competitive pan–epidermal growth factor receptor (EGFR) family kinase inhibitors. Biochemical analysis confirmed the existence of distinct compound-induced TRIB2 conformations, and a compound screen identified known EGFR family inhibitors that stabilize or destabilize TRIB2 in vitro. TRIB2-binding compounds included the clinically used breast cancer therapeutic lapatinib (marketed as Tykerb) ( 41 ) and the U.S. Food and Drug Administration–approved irreversible electrophilic covalent inhibitors afatinib (Giotrif) ( 42 , 43 ) and neratinib (Nerlynx) ( 44 , 45 ). In the case of the latter two destabilizing agents, binding led to uncoupling of the pseudokinase domain from its own C-terminal tail. Consistently, afatinib exposure led to rapid TRIB2 degradation in cells, driven by an interaction with the Cys-rich pseudokinase domain, which interfered with AKT signaling and decreased cell survival in a TRIB2-expressing leukemia model. The availability of target-validated compounds that act as rapid TRIB2 pseudokinase down-regulators through a direct effect on the pseudokinase represents a new way to evaluate TRIB2 physiology and cell signaling. It might also have a broader impact on the rapidly developing pseudoenzyme field ( 46 ), where the concept of pseudokinase destabilization or elimination by targeted kinase “inhibitors” ( 7 ) has a number of potentially useful applications.

Tribbles pseudokinases are implicated in many physiological signaling pathways, often in the context of protein stability, but also through regulation of key modules, such as the canonical AKT pathway ( 32 ). TRIB2 is also implicated in the etiology of human cancers, including leukemia, melanoma, and lung and liver cancers ( 33 ). In particular, TRIB2 is a potential drug target in subsets of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), which are in urgent need of targeted therapeutics to help treat untargeted or drug-resistant patient populations ( 34 ). TRIB2 protein abundance has also been linked to drug resistance mechanisms, where an ability to modulate the prosurvival AKT signaling module underlies a central regulatory role in cell proliferation, differentiation, metabolism, and apoptosis ( 32 , 35 – 39 ).

A long-standing goal in cancer research is drug-induced degradation of oncogenic proteins. Progress toward this objective has been transformed by the synthesis of proteolysis-targeting chimeras (PROTACs), which induce proteasome-dependent degradation of their targets. Multifunctional small-molecule PROTACS often have protein-binding regions derived from kinase inhibitors ( 22 , 23 ), and multiple classes of non-PROTAC kinase inhibitors also induce kinase target degradation, although typically at higher (micromolar) concentrations than those required for enzymatic inhibition ( 7 ). Recent reports also disclose classes of covalent compounds that bind and disable cysteine (Cys)–containing small guanine nucleotide–binding proteins such as mutant human RAS, permitting covalent inactivation of this previously “undruggable” oncoprotein ( 24 , 25 ). Cys residues are widespread and highly conserved in kinases ( 26 ), and the conservation of Cys residues both inside and outside the catalytic domain provides kinome-wide opportunities for exploitation using chemical biology ( 27 ). In this context, covalent targeting methodologies involving compound-accessible Cys residues in kinases ( 8 , 28 – 31 ) and pseudokinases ( 3 ) have attracted substantial attention for small-molecule design, due to the potential for gains in target specificity and durability of responses, combined with tractability in experimental systems.

The three human TRIB pseudokinases (TRIB1, TRIB2, and TRIB3) and the related pseudokinase STK40 (serine/threonine kinase 40, also known as SgK495) are homologs of the Drosophila melanogaster pseudokinase termed Tribbles, which controls ovarian border cell and neuronal stem cell physiology in flies ( 13 , 14 ). These proteins contain a catalytically impaired pseudokinase domain. Adaptations in the pseudokinase fold, including a highly unusual αC-helix, are thought to support a competitive regulatory interaction in cis with a unique C-terminal tail DQLVP motif ( 15 – 17 ). Through a still obscure mechanism, TRIB and STK40 function as adaptor proteins that recruit ubiquitin E3 ligases, such as constitutive photomorphogenesis protein 1 homolog, through interaction with the conserved C-tail peptide ( 15 , 17 ), which is also required for signaling and cellular transformation ( 18 ). Mechanistically, the signaling outputs of Tribbles proteins are controlled through the ubiquitylation and subsequent proteasomal destruction of Tribbles “pseudosubstrates”; in vertebrates, these include the transcription factor CCAAT/enhancer binding protein α (C/EBPα), the cell cycle–associated phosphatase CDC25C, and the enzyme acetyl coenzyme A carboxylase ( 19 – 21 ).

The human protein kinome encodes ~60 protein pseudokinases, which lack at least one conventional catalytic residue but often control rate-limiting signaling outputs within cellular networks ( 1 ). Like canonical kinases, pseudokinases drive conformation-dependent signaling associated with both physiology and disease ( 2 , 3 ). The human “pseudokinome” includes cancer-associated signaling proteins such as human epidermal growth factor receptor 3 (HER3), Janus kinase 2 (JAK2; JH2 domain), and Tribbles 2 (TRIB2), which have received much less attention than their conventional, catalytically active counterparts even though pseudokinase domains represent rational targets for drug discovery ( 4 ). Discovering or repurposing biologically and/or clinically active compounds that target atypical conformations of canonical kinases or pseudokinases is an area of active research ( 2 , 3 , 5 – 9 ). Moreover, the burgeoning pseudokinase field is strongly placed to benefit from the decades of research undertaken on canonical protein kinases, which has seen the approval of more than 40 kinase inhibitors for human cancer and inflammatory diseases ( 10 , 11 ). This includes understanding how adenosine triphosphate (ATP)–competitive, allosteric, or covalent inhibitors might influence pseudokinase-based signaling mechanisms that are relevant to health and disease ( 3 , 12 ).

RESULTS

Analysis of human TRIB2 using a thermal stability assay Human TRIB2 differs from TRIB1, TRIB3, and STK40 in the pseudokinase domain because of a Cys-rich region at the end of the β3-Lys–containing motif leading into the truncated αC-helix in the N-lobe (Fig. 1A, top). We developed a differential scanning fluorimetry (DSF) assay (47–49) to examine thermal stability of full-length (1 to 343) His-tagged TRIB2 proteins and compared it to either full-length adenosine 3′,5′-monophosphate (cAMP)–dependent protein kinase (PKAc) catalytic subunit, which is a model for canonical kinases, or full-length C104Y TRIB2, in which Cys104 was replaced with the Tyr residue conserved in human TRIB1 and TRIB3 (Fig. 1A). Proteins were purified to homogeneity (Fig. 1A, bottom), and thermal stability based on unfolding profiles was determined for each protein (reported as a T m value, Fig. 1B). As previously demonstrated (40), TRIB2 (T m = ~39°C) was much less thermostable than the canonical protein kinase (PKA, T m = 46.3°C). Remarkably, the C104Y single substitution induced stabilization of TRIB2, with the T m value increasing to ~49°C, comparable to that of human TRIB1 (15), suggesting an important structural role for this unique Cys residue in TRIB2 (un)folding dynamics. To confirm that recombinant TRIB2 binds to a known physiological target, we demonstrated that glutathione S-transferase (GST)–tagged TRIB2 interacted preferentially with catalytically inactive (non–Thr308-phosphorylated) AKT1 in vitro (Fig. 1C). Consistent with a functional regulatory interaction between TRIB2 and AKT in cells (50), transient overexpression of tetracycline (TET)–inducible FLAG-tagged TRIB2 in HeLa cells led to a large increase in endogenous AKT phosphorylation at the hydrophobic motif (Ser473, Fig. 1D), an established marker for AKT catalytic activity and generation of a downstream cellular antiapoptotic signal (51). Fig. 1 Full-length TRIB2 is a target for protein kinase inhibitors in vitro. (A) Top: Sequence alignment of human TRIB2, TRIB1, TRIB3, and STK40/SgK495, highlighting Cys-rich residues (numbered in red) in the TRIB2 pseudokinase domain. Bottom: Blot of the recombinant proteins used for in vitro analysis. The indicated purified proteins (5 μg each) were resolved by SDS–polyacrylamide gel electrophoresis (SDS-PAGE). WT, wild-type; BSA, bovine serum albumin. (B) Thermal denaturation profiles of recombinant proteins. A representative unfolding profile is shown. T m values (±SD) were obtained from three separate fluorescence profiling experiments, each point assayed in duplicate. (C) The ability of GST-TRIB2 to interact with active [3-phosphoinositide-dependent protein kinase 1 (PDK1)–phosphorylated] or inactive (non–PDK1-phosphorylated) S473D AKT1 was assessed by glutathione-Sepharose pulldown followed by immunoblotting. Left: “Master-mix” input. (D) Transient transfection of TET-inducible FLAG-TRIB2 leads to increased AKT phosphorylation on Ser473. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (E) TRIB2 DSF screen using PKIS. His-TRIB2 (5 μM) was used for all DSF analysis. ΔT m values were calculated for each compound (N = 2). Scattergraph of data highlights a wide variety of compounds that either stabilize or destabilize TRIB2 in vitro. Cutoff values of >+3.5°C and <−2°C were used to designate hits. (F) Comparative DSF analysis of clinical and preclinical kinase inhibitors as potential TRIB2-binding compounds. LAP, lapatinib; TAK, TAK-285; AFA, afatinib; NER, neratinib; OSI, osimertinib; IBR, ibrutinib; ERL, erlotinib; GEF, gefitinib. (G) Dose-dependent analysis of thermal shifts induced by clinical TRIB2-binding compounds. Compounds were tested at 5, 10, 20, 40, 80, and 160 μM. (H) Profiling of TRIB2 and C104Y with selected inhibitors by DSF.

A DSF screen for TRIB2-binding compounds using a kinase inhibitor library The ability of full-length recombinant human TRIB2 to bind to ATP in the presence of EDTA (40, 48) confirms that a vestigial nucleotide-binding site is present within the pseudokinase domain. Moreover, our previous work established that an analog-sensitive (F190G) TRIB2 variant could be stabilized by bulky pyrimidine analogs in vitro (40). To discover drug-like compounds for WT (full-length) TRIB2, we screened the Published Kinase Inhibitor Set (PKIS), a collection of high-quality class-annotated kinase inhibitors (52). We enforced cutoff values of ~ΔT m = <−2°C and >+3.5°C (therefore eliminating ~97% of the library) to define “hit” compounds that had the ability to destabilize or stabilize TRIB2 in a thermal stability assay at a 1:4 TRIB2/compound molar ratio (Fig. 1E and table S1). The top “stabilizing” compound identified was GW693881A, a dual EGFR and HER2 thienopyrimidine inhibitor with a ΔT m of +4.7°C. The top “destabilizing” compound was GW804482X, a thiophene polo-like kinase (PLK) inhibitor that induced a ΔT m of −3.4°C (Fig. 1E, red symbols). Most of the top stabilizing and destabilizing compounds belonged to well-known ATP-competitive pyrimidine or quinazoline EGFR family chemotypes (fig. S1) (53–55), suggesting broad structural cross-reactivity between TRIB2 and a compound-binding EGFR and HER2 conformation. To build upon these findings, we screened a larger panel of known dual EGFR and HER2 inhibitors (fig. S2) and established that the clinical type I EGFR family inhibitors TAK-285 and lapatinib also stabilized TRIB2 in vitro (Fig. 1F). The ATP-competitive covalent EGFR family inhibitors afatinib, neratinib, and osimertinib (but not the unrelated covalent Bruton’s tyrosine kinase inhibitor ibrutinib or the type I EGFR-specific inhibitors erlotinib and gefitinib) destabilized TRIB2, similar to the PLK inhibitor GW804482X (Fig. 1F) and the dual EGFR family inhibitor GW569530A (fig. S1). As expected (15), purified TRIB1 (fig. S3A) was more thermostable than TRIB2 in the absence of kinase inhibitors (fig. S3B). However, it was not destabilized by afatinib, neratinib, or osimertinib, although like TRIB2, destabilization was evident in the presence of GW804482X (fig. S3C), in agreement with independent findings (56). Compound effects were caused through pseudokinase targeting in the thermal shift assay, because no shift was elicited when the canonical kinase PKAc was compared in a side-by-side counterscreen, with dasatinib as a positive control (fig. S3D). The preclinical PLK inhibitors BI2536 and BI6727 (volasertib) had no discernible effects on TRIB2 stability in this assay, in contrast to the chemically distinct thiophene PLK inhibitor GW804482X (Fig. 1F). Afatinib, neratinib, and osimertinib are covalent (type IV) inhibitors of EGFR family tyrosine kinases, interacting irreversibly with a conserved Cys residue in the canonical ATP-binding site (57). The stabilization of TRIB2 by lapatinib, and the destabilization of TRIB2 by all three covalent inhibitors, occurred in a dose-dependent manner (Fig. 1G and fig. S4). A C104Y TRIB2 mutant was no longer destabilized by either afatinib or neratinib but remained “sensitive” to lapatinib TAK-285 and GW804482X on the basis of thermal protection (Fig. 1H and fig. S5A). None of these latter compounds contain the electrophilic “warhead” required for covalent interactions (fig. S2). Elution profiles from Superdex 200 were identical for WT and C104Y TRIB2 (fig. S5B), confirming that both proteins were monomeric species in solution, with an estimated relative molecular mass (M r ) of 45.3 kDa. C104Y TRIB2 was also insensitive to thermal shift in the presence of ATP and EDTA (Fig. 1H and fig. S5C). These data suggest that the amino acid identity at TRIB2 position 104 is likely important for both ATP binding and interaction with covalent kinase inhibitors that induce TRIB2 destabilization in vitro.

Mechanistic analysis of TRIB2 structural stability and TRIB2 compound binding Structural analysis of TRIB1 by x-ray crystallography and small-angle x-ray scattering led to the proposal of an in cis self-assembly model, whereby the unique C-terminal tail region, which contains the conserved “DQLVP” motif, binds directly to the pseudokinase domain adjacent to the short αC-helix of TRIB1 (15, 16, 56). To investigate whether this mechanism is also relevant in TRIB2, we generated a series of truncated proteins. These lacked either the N-terminal extension, which was predicted to be disordered by both I-TASSER (Iterative Threading ASSEmbly Refinement) (58) and VSL2 (59), the C-terminal tail, or both N- and C-terminal regions. We also generated a triple-point mutant in which the DQLVP tail motif, which is required for TRIB1 and TRIB2 cellular transformation in vivo (18), was mutated to a nonfunctional “AQLAA” sequence (Fig. 2A). Both full-length (1 to 343) TRIB2 and TRIB2 lacking the N-terminal domain (TRIB2 54 to 343) exhibited similar T m values of 39° to 40°C (Fig. 2B). In contrast, deletion of the C-tail (TRIB2 1 to 318) changed TRIB2 stability, with T m values falling to ~37°C, diagnostic of a destabilized TRIB2 conformation (Fig. 2B). Mutation of DQLVP to AQLAA further destabilized TRIB2, leading to a T m value of ~36°C (Fig. 2B). Using this panel of recombinant TRIB2 proteins, we measured the relative effects of kinase inhibitors on TRIB2 stability. Consistent with a lack of effect on compound interactions, deletion of the TRIB2 N-terminal region had no effect on ΔT m values induced by any compound. However, removal of the C-tail region (54 to 318 and 1 to 318 mutants) abolished afatinib- and neratinib-induced TRIB2 destabilization but had a negligible effect on GW804482X binding (Fig. 2C). Consistently, destabilization by afatinib was also completely abolished in the AQLAA triple mutant, whereas neratinib effects were reduced by >50%. Notably, neither the destabilizing effect of GW804482X nor the stabilizing effects of lapatinib or TAK-285 differed between any of the TRIB2 proteins evaluated. These results suggest a very similar destabilizing mechanism induced by covalent EGFR family compounds via displacement of the TRIB2 C-tail, which is a unique feature of Tribbles pseudokinases (13, 15). Fig. 2 TRIB2 thermal stability is modulated through Cys binding to covalent inhibitors. (A) Top: Schematic cartoon of TRIB2 with domain boundaries numbered and cysteine residues highlighted (red). Bottom: SDS-PAGE of 5-μg recombinant TRIB2 proteins. (B) Thermal denaturation profiles of 5 μM WT-TRIB2 (amino acids 1 to 343), three truncated variants, and an AQLAA triple-point mutant. Representative curves for each protein and average T m values (±SD) are shown, calculated from N = 3 experiments. (C) Thermal shift analysis of TRIB2 deletion and AQLAA proteins measured in the presence of a panel of compounds (20 μM). The change in T m value (ΔT m ) is reported from N = 3 experiments, each performed in triplicate. (D) Thermal denaturation profiles for purified TRIB2 and C96S, C104S, and C96/104S proteins. (E) Thermal shift analysis of TRIB2 Cys-mutated proteins measured in the presence of a panel of compounds (20 μM). The change in T m value (ΔT m ) is reported from N = 3 experiments. To evaluate whether afatinib targeted unique Cys residues in the TRIB2 pseudokinase domain (Fig. 1A), we performed mass spectrometry (MS) analysis to evaluate protein modification. As detailed in fig. S6A, incubation of TRIB2 with a fivefold molar excess of afatinib led to covalent interaction with Cys96 in TRIB2. A doubly charged chymotryptic product ion representing the TRIB2-derived DISC96Y:afatinib peptide adduct at mass/charge ratio (m/z) 543.2 (fig. S6A) and the isotopic ratios of the 35Cl- or 37Cl-containing peptide ions unequivocally confirmed Cys96 as a site of TRIB2 binding (fig. S6B). Having confirmed an intact mass for recombinant full-length TRIB2 of 43,587.09 Da, very similar to the predicted mass of 43,587.22 Da (fig. S6C), we were also able to ascertain that preincubation with afatinib generated covalent adducts containing predominantly either one or two molecules of afatinib. There was also some evidence for tri- and tetra-modified TRIB2 adducts (fig. S6D). We next examined afatinib interaction with TRIB2 by MicroScale Thermophoresis (MST), a biophysical technique for quantification of reversible biomolecular interactions (60). This revealed an interaction between fluorescent nitrilotriacetic acid–coupled His-TRIB2 and two compounds, which could be fitted to reversible binding with dissociation constant (K d ) values of ~16 μM for afatinib and ~20 μM for TAK-285 (fig. S7, A and B). A sub-micromolar interaction between myeloid cell leukemia–1 (MCL-1) and A1210477 served as a positive control (61). In agreement with MS data (fig. S6), we therefore propose initial (reversible) binding of ATP-competitive afatinib (62, 63), before subsequent formation of a covalent adduct (or adducts) with the TRIB2 pseudokinase domain. We also evaluated the potential interaction of afatinib and neratinib using TRIB2 modeled on the C/EBP-bound “SLE-in” conformation from TRIB1 (13, 56) and compared it to the known afatinib target EGFR (in a “DFG-in” conformation) using AutoDock Vina (64). Docking of covalent inhibitors revealed a putative binding pocket formed by residues from the vestigial TRIB2 C-helix, including Cys96, and the β3 strand (fig. S8A). Afatinib and neratinib dock in a structurally similar pose in which the enamine β carbon orients toward the sulfhydryl group of Cys96. This docking pose, however, is distinct from the afatinib-bound crystal structure of EGFR, wherein Cys797 is located in the D-helix. We performed molecular dynamics simulations (fig. S8B) on the docked complexes to assess the feasibility of the binding poses. Both afatinib and neratinib remained stably bound to TRIB2 for nearly 17 ns, suggesting that the binding poses are favorable and feasible (65). The enamine β carbon remained within 3 to 5 Å of the Cys96 sulfur atom for the majority of the simulation. Given the rapid nature of the thiol-ene reaction coupled with the proximity of reactants, we believe that this time frame offers sufficient time for the formation of a covalent bond (66), which we confirmed by MS analysis. To validate the biochemical importance of Cys residues for afatinib binding, we next examined interaction with TRIB2 in which two Cys amino acids were mutated to non–thiol-containing Ser residues. Individual or combined mutation of Cys96 and Cys104 to a Ser residue had no effect on the thermal stability (T m ) of the purified TRIB2 proteins (Fig. 2D), in contrast to the highly stabilizing effect of a Tyr at position 104 (Fig. 1B and fig. S5C). However, individual or joint mutation of Cys96 and Cys104 to Ser severely blunted the destabilizing effect of afatinib and neratinib on TRIB2, in contrast to the noncovalent TRIB2 compound GW804482X or the EGFR family stabilizing compounds lapatinib and TAK-285 (Fig. 2E). Together, our findings confirm that these covalent compounds elicit effects on TRIB2 through Cys96 and/or Cys104.