Identifying a selective inhibitor of cell motility

We selected flavonoids as a chemical scaffold to advance probe synthesis because they exert a wide range of biological effects6. We began with 4′,5,7-trihydroxyisoflavone (genistein) as our starting point because of its known anti-motility properties. We previously demonstrated that nanomolar concentrations of genistein inhibit human prostate cancer (PCa) cell invasion in vitro7, metastasis in a murine orthotopic model8, and in the context of a prospective human trial that it downregulates matrix metalloproteinase 2 (MMP-2) expression in prostate tissue9. While its diverse spectrum of biological effects render it unusable as a selective and potent biological probe, these same properties maximize its potential to selectively probe a wide spectrum of bioactive sites upon chemical diversification.

We developed a series of related molecular probes through phenotypically driven structure activity relationship studies, specifically through chemical modification of the genistein structure (aromatic substitution and ring saturation). These compounds were advanced by iterative selection for inhibition of human PCa cell invasion (Fig. 1a and Supplementary Note 1 and Supplementary Fig. 1). A major parallel goal was deselection for inhibition of cell growth (an indicator of off-target effects). Knowing that genistein has estrogenic action, and guided by the crystal structure of genistein bound to estrogen receptor (ER)10, we also de-selected for ER-binding. Through this strategy, (±)-3(4-fluorophenyl)chroman-4-one (KBU2046), a halogen-substituted isoflavanone, was discovered (Fig. 1a).

Fig. 1 KBU2046 selectively inhibits cell motility. a Schematic flow of probe synthesis and development strategy. b Cell invasion. Human prostate metastatic cells (PC3, PC3-M), and HPV-transformed normal (1532NPTX, 1542NPTX) and primary cancer (1532CPTX, 1542CPTX) cells, were treated with 10 µM genistein (G), KBU2046 (46), or vehicle (CO), and after 3 days, cell invasion was measured. Values are mean ± SEM of a single experiment in replicates of N = 4, with similar findings in multiple separate experiments (also N = 4). c Single-cell migration. Cell migration was measured after treatment for 3 days with 10 µM KBU2046 or vehicle (control). Values are mean ± SEM of a single experiment in replicates of N = 24, with similar findings in a separate experiment (also N = 24). *Denotes Student’s t-test P value <0.05, compared to controls. d Human cord blood hematopoietic stem cell colony formation assay. Values are the mean ± SD number of total, CFU-GM, CFU-GEMM, or BFU-E colonies at 14 days after treatment with KBU2046, from a single experiment in replicates of N = 2. e Induction of estrogen-responsive genes. Values are the mean ± SD of a single experiment, with similar results seen in a separate experiment, both in replicates of N = 2 Full size image

KBU2046 inhibits cell invasion with efficacy equal-to-or-greater-than that of genistein for human prostate cells, including normal prostate epithelial cells, as well as primary and metastatic PCa cells (Fig. 1b). Cell migration is a major determinant of cell invasion11, and KBU2046 inhibited the migration of human prostate, breast, colon, and lung cancer cells (Fig. 1c). Importantly, KBU2046 had high selectivity in cellular assays. It was not toxic to human prostate cells (Table 1), to human bone marrow stem cells (Fig. 1d), nor to cells in the NCI-60 cell line panel (Supplementary Fig. 2). Bone marrow toxicity is induced by a wide spectrum of therapeutic agents, and is frequently a dose-limiting toxicity for anti-cancer agents. Furthermore, in estrogen-responsive human breast cancer MCF-7 cells, KBU2046 did not activate estrogen-responsive genes (Fig. 1e and Supplementary Fig. 3).

Table 1 Effect of KBU2046 and genistein on human prostate cell viability Full size table

KBU2046 inhibits metastasis and prolongs survival

Because metastasis is a systemic process, effective small molecule probes must operate at the systemic level. We designed the probe to contain chemical properties known to be associated with systemically active small molecules (Supplementary Fig. 4). Employing a murine orthotopic implantation model of human PCa previously characterized by us8, KBU2046 was shown to significantly decrease metastasis in a dose-dependent manner by up to 92%, at plasma concentrations of 1.1–24 nM (Fig. 2a, b). Comprehensive characterization of KBU2046 pharmacokinetics demonstrated maintenance of plasma concentrations >24 nM for 9.3 h after a single oral dose, and allowed for characterization of pharmacokinetic parameters (Fig. 2c and Supplementary Fig. 5). At the systemic level, KBU2046 was a highly selective inhibitor of metastasis. Comprehensive analysis of primary tumor growth, animal behavior, weight, histologic examination of multiple organs, and serum chemistry profiling failed to identify KBU2046-associated off-target effects (Supplementary Figs. 6, 7).

Fig. 2 KBU2046 inhibits cancer metastasis and prolongs life. a, b Inhibition of PCa metastasis. Cohorts of N = 12 athymic mice bearing human PCa PC3-M cell orthotopic implants (a), or of N = 5 non-tumor bearing athymic mice (b), were treated with KBU2046 incorporated into chow, and resultant lung metastasis (a) or plasma KBU2046 concentration (b) measured. Values are the mean ± SEM. The relationship between dose and metastasis was evaluated by two-sided ANOVA (a). c Comprehensive characterization of KBU2046 pharmacokinetics. CD1 mice were dosed with 100 mg KBU2046/kg via oral gavage or intravenous injection (iv), and blood collected at the indicated time points (data from mice dosed at 25 mg/kg are in Supplementary Fig. 6, and corroborate 100 mg/kg findings). For each route and time point, N = 3 mice were sampled. Individual data points are the resultant plasma concentrations from individual mice, and are the mean of N = 2 measurements. The dotted horizontal line denotes a concentration of 24 nM, which was the concentration of KBU2046 measured in the blood of mice whose metastasis were suppressed by 92% (a, b). d Prolongation of survival in BCa-bearing mice. Mice were orthotopically implanted with human breast cancer LM2-4H2N cells, the resultant primary tumors resected, and adjuvant treatment begun with KBU2046 by daily oral gavage five times per week. The survival of N = 6 mice receiving vehicle was compared to that of N = 6 mice receiving 25 mg/kg KBU2046 by the log-rank (Mantel-Cox) test Full size image

Recognizing the established link between metastasis and decreased survival in humans, we evaluated KBU2046’s impact on survival. The orthotopic PCa model exhibits tumor growth around the urogenital tract, inhibiting renal function and precluding assessment of the impact of metastatic burden on survival. However, orthotopic implantation of human breast cancer cells, followed by surgical removal of the resultant primary tumor, provides a murine model wherein survival is dictated by metastatic burden12. KBU2046 significantly prolonged the survival of mice treated in a post-surgery adjuvant setting (Fig. 2d).

If KBU2046 were inhibiting metastasis through inhibition of cell motility, then it should have little to no effect upon the metastatic process once cells have implanted into distant organs. Recognizing that skeletal metastases are a major clinical problem with PCa, further assessment of this paradigm was pursued with an established PCa bone metastasis model13. PC3 luciferase tagged (PC3-luc) cells were delivered by ultrasound-guided intracardiac (IC) injection and metastatic outgrowth monitored weekly via IVIS imaging (Fig. 3a and Supplementary Fig. 8). Compared to control mice, the pre-cohort of mice (KBU2046 treatment starts 3 days prior to IC injection and continuing through the end of the experiment) experienced a significant decrease in total metastatic burden, as well as decreased metastasis to the mandible (for which this model is designed) (Fig. 3b, c). In contrast, with the post-cohort of mice, metastasis to the total body as well as to the jaw do not differ from control mice. With the post-cohort of mice, cells are given 3 days post IC injection to invade into distant organ sites before treatment is begun, with treatment then continuing through the end of the experiment. In the Pre7Stop cohort of mice, treatment starts 3 days prior to IC injection, continues through day 7 post IC injection and is not given for the remaining 3 weeks of the experiment. Findings in this cohort of mice suggest an intermediate outcome between that of pre- and post-cohorts. Specifically, total body metastasis is significantly decreased in the Pre7Stop cohort, compared to both control and post-cohorts. While jaw metastasis is significantly decreased compared to control, it is not significantly decreased compared to the post-cohort of mice, yet the average value is below that of post-mice and is approaching that of the pre-cohort of mice. Degradation of the mandible was quantified with computed tomography in pre-, post-, and control cohorts, demonstrating decreased destruction of bone in the pre-cohort of animals (Fig. 3d, e).

Fig. 3 KBU2046 inhibits bone destruction. a Treatment schema. Athymic mice were given intracardiac (IC) injections of PC3-luc cells on day 0 under ultrasound guidance, and underwent weekly IVIS imaging starting 7 days post injection. Cohorts of N = 20 control, N = 20 pre (treatment from 3 days prior to IC injection through end of the experiment), N = 10 Pre7Stop (treatment from 3 days prior to IC injection through 7 days post IC injection), and N = 10 post-treatment (treatment from 3 days post IC injection through end of the experiment) mice were dosed with 80 mg/kg KBU2046 daily by oral gavage and mock-treated with vehicle all other times. Whole-body (b) and mandible (c) flux are depicted, as determined from weekly IVIS imaging. d At week 4 post injection (i.e., at the end of the experiment), CT scans were performed on control, pre-, and post-treatment cohorts, and mandibular destruction quantified. e Representative images of pre and control mice are depicted. Arrows denote areas of bone destruction in controls, and corresponding areas in the pre mouse. Student’s t-test (b, c) and Fisher’s exact test (d) P values between the denoted cohorts are shown Full size image

KBU2046 induces changes in HSP90β phosphorylation

With the aforementioned positive phenotypic cellular and animal studies, we sought to identify the molecular basis for KBU2046’s biological action. Our initial investigations were guided by our prior demonstration that low nanomolar concentrations of genistein inhibited the kinase activity of mitogen-activated protein kinase 4 (MKK4/MAP2K4/MEK4)9, in turn inhibiting downstream phosphorylation of p38 MAPK7 and of heat-shock protein 27 (HSP27)14. This translated into inhibition of MMP-2 expression and cell invasion in vitro, inhibition of human PCa metastasis in mice8 and decreased MMP-2 expression in human prostate tissue9. In contrast to genistein, KBU2046 did not bind to MKK4 nor inhibit its kinase activity in vitro, and it did not inhibit downstream phosphorylation of p38 MAPK or of HSP27 in cells (Supplementary Fig. 9). This finding, while surprising, demonstrates that our chemical probe strategy de-selected for inhibition of the MKK4 signaling axis. Importantly, this provides a measure of the unbiased nature of our chemical probe strategy.

Seeking to identify KBU2046’s biological target(s), we pursued alternative methods. The KinomeView® panel of antibodies (Cell Signaling Technology, Inc.) detect established protein phosphorylation motifs, and were used to probe for KBU2046-induced changes in protein phosphorylation (Fig. 4a and Supplementary Fig. 10). We prioritized phosphoprotein changes that met the following criteria: were induced in cells in vitro as well as in tumors of treated mice (from Fig. 2a), that counteracted transforming growth factor β (TGFβ)-induced effects, and that were reproducible. TGFβ is ubiquitous in vivo, is known to increase PCa cell invasion7, and KBU2046’s anti-invasion efficacy remains in spite of TGFβ-stimulated increases in cell invasion (Supplementary Fig. 11). Genistein was evaluated under identical treatment conditions for comparison. Genistein’s many pharmacologic effects induced widespread changes in protein phosphorylation (Supplementary Fig. 10). In contrast, KBU2046 induced only a single change that met our pre-specified criteria, i.e., a decrease in intensity of an 83 kDa protein band (blue arrow in Fig. 4a). In tumors of treated animals, KBU2046 had this same effect on this 83 kDa protein band (green arrow in Supplementary Fig. 10a). The high molecular selectivity of KBU2046 was further supported by its failure to inhibit over 400 different protein kinases and 20 phosphatases examined, in three different in vitro assay systems (Supplementary Note 2).

Fig. 4 KBU2046 decreases phosphorylation of HSP90β. a Probing for KBU2046-induced changes in protein phosphorylation. PC3-M or PC3 cells were pre-treated with 10 µM KBU2046 for 3 days, then with ±TGFβ and the resultant cell lysate probed for changes in protein phosphorylation with the KinomeView® assay. The depicted western blot utilizes KinomeView® phospho-motif antibody, BL4176; the blue arrow denotes an 83 kDa band whose phosphorylation is inhibited by KBU2046 (see Supplementary Fig. 10 for complete KinomeView® assay screening data). b Proteomic analysis. PC3 cells were pre-treated with KBU2046 or vehicle, then with TGFβ, proteins from the resultant cell lysate were immunoprecipitated with BL4176, and HSP90β was identified by LC-MS/MS analysis (see Supplementary Fig. 12 for expanded proteomic assay data). The phospho-motif recognized by the antibody is underlined; S*—denotes Ser226, whose phosphorylation is decreased by KBU2046. c, d The phospho-mimetic changes in HSP90β Ser226 structure regulate human PCa cell invasion and KBU2046 efficacy. PC3-M cells were transfected with S226A-, S226D-, or WT-HSP90β, or empty vector (VC), treated with KBU2046 or vehicle, and cell invasion measured. Values are the mean ± SEM of a representative experiment of multiple experiments (all in replicates of N = 3); *denotes t-test P value <0.05 between bracketed conditions, or compared to VC Full size image

The 83 kDa protein was identified by pretreating PC3 cells with KBU2046 or vehicle control, treating with TGFβ and performing LC-MS/MS analysis on proteins pulled down by the KinomeView® antibody used in Fig. 4a. Resultant data were analyzed with the SEQUEST/Sorcerer data analysis suite, and proteins further selected based upon predetermined parameters (Supplementary Fig. 12). This approach yielded a single protein, HSP90β, and indicated that KBU2046 decreased the abundance of phosphorylated Ser226 on HSP90β by 6.6-fold (Fig. 4b and Supplementary Fig. 12).

The (S226A)-HSP90β construct lacks a Ser226 residue, precluding phosphorylation at that site, represents a constitutive inactive mimic, and mimics the effect of KBU246 on that residue (i.e., dephosphorylation). As expected, transfection of cells with (S226A)-HSP90β inhibited cell invasion, compared to vector control (VC) transfected cells (Fig. 4c). Further, if KBU2046 were exerting efficacy by inhibiting phosphorylation of the Ser226 residue, then removal of that residue should, by definition, preclude additional efficacy. This is exactly what is observed: in (S226A)-HSP90β transfected cells, KBU2046 does not further inhibit cell invasion, while it significantly inhibits invasion in VC cells (Fig. 4c and Supplementary Fig. 13a). The selectivity of HSP90β in mediating KBU2046 efficacy was further supported by demonstrating that small interfering RNA (siRNA)-mediated HSP90β knockdown inhibited cell invasion and abrogated KBU2046 efficacy (Supplementary Fig. 13b–d). HSP90β-specific siRNA did not knockdown HSP90α (Supplementary Fig. 13b). Conversely, the pseudophosphorylated (S226D)-HSP90β construct contains a residue that provides a biological mimic of phosphorylated Ser226, and as such constitutes a constitutively active mutant. As expected, cells transfected with (S226D)-HSP90β were more invasive than VC cells (Fig. 4d). Recognizing that pseudophosphorylated constructs only serve to mimic activated wild-type protein, it was not surprising that (S226D)-HSP90β cells were not as invasive as WT-HSP90β cells. More importantly, if KBU2046 were exerting efficacy by inhibiting phosphorylation of the Ser226 residue, then the presence of a phospho-mimic residue should, by definition, decrease additional efficacy. This is exactly what is observed: in (S226D)-HSP90β transfected cells, KBU2046 did not significantly inhibit cell invasion, while it significantly inhibited invasion in both VC and WT-HSP90β cells (Fig. 4d and Supplementary Fig. 13). These findings demonstrate that changes in the phosphorylation status of Ser226 on HSP90β can be altered by a small molecule, and appear to be associated with selective inhibition of cancer cell motility by KBU2046.

KBU2046 selectively disrupts heterocomplex function

KBU2046’s effect upon HSP90β function is completely different from that of classical HSP90 inhibitors. The latter induce cytotoxicity and work by binding directly to HSP90, thereby inhibiting its enzyme activity, in turn affecting the function of large numbers of cellular kinases and other client proteins15. In contrast, KBU2046 was not cytotoxic and its effects on protein phosphorylation were highly specific, demonstrating a lack of effects on kinase function. HSP90β is part of a large multiprotein chaperone complex whose function involves binding a large but specific set of regulatory proteins. We reasoned that KBU2046 was changing the signature of bound client proteins, that the change was highly selective in terms of number of affected proteins, and that it was highly specific for proteins that regulate cell motility.

CDC37 is a co-chaperone that mediates the binding of over 350 client proteins to HSP90β, including over 190 kinases16. CDC37 is a flexible arm-like structure (protein data bank (PDB) ID: 2WOG), is highly dynamic17, enables binding of large numbers of kinases, defines their positioning and thereby their potential to affect HSP90β phosphorylation status. We reasoned that KBU2046 was binding to either CDC37 or HSP90β, that this altered the function of the CDC37/HSP90β heterocomplex resulting in a change in the spectrum of bound client kinase proteins, that changes were highly selective and that this altered binding spectrum would in turn be responsible for KBU2046’s effects upon cell motility.

There was no evidence of KBU2046 binding to either CDC37 or HSP90β by biophysical methods, inclusive of isothermal titration calorimetry, fluorescence-based thermal shift assay, biolayer interferiometry, or by dynamic light scattering, nor by the biochemical method of drug affinity responsive target stability (DARTS) assay (Supplementary Fig. 14). DARTS provides a sensitive measure of ligand-induced changes in protein structure and dynamics by measuring the ability of a ligand to protect its target from protease digestion18. Although KBU2046 did not bind CDC37 or HSP90β individually, because CDC37 and HSP90β associate to form a heterocomplex17, we went on to combine CDC37 and HSP90β in a DARTS assay, demonstrating that KBU2046 protected both proteins from digestion (Fig. 5a). The intensity of the CDC37 band increased, that of the HSP90β degradation product decreased, and both effects were statistically significant, concentration-dependent, and were evident at 10 nM. Further, the high selectivity of KBU2046 for protein binding was additionally supported by synthesizing a biotin chemical linker to KBU2046, demonstrating that it retained biological activity, that it bound to intact cells (i.e., under physiological conditions of CDC37/HSP90β heterocomplex formation), and that it failed to bind to an array of over 9000 human proteins (Supplementary Fig. 15). Together, these findings demonstrate that KBU2046 will not bind to either CDC37 or HSP9Oβ, but that it will only bind when both proteins are present and able to form heterocomplexes. Further, all findings also indicate that KBU2046 is not acting as a classical HSP90 inhibitor. Classical HSP90 inhibitors bind isolated HSP90, without the need for co-chaperones being present, are characterized by their cytotoxic effects, are systemically toxic, particularly to the liver, and broadly inhibit client kinase protein binding, thereby exerting widespread effects upon cellular signaling and affecting a wide array of cellular processes15. In contrast, KBU2046 exhibits a complete lack of cellular cyctotoxicity and systemic toxicity, everts highly specific effects in both molecular-based protein phosphorylation, and cellular-based functional assays and will not bind HSP90 in isolation.

Fig. 5 KBU2046 stabilizes CDC37/HSP90β heterocomplexes. a KBU2046 stabilizes HSP90β/CDC37 heterocomplexes in a DARTS assay. Equimolar amounts of HSP90β and CDC37 protein were pre-incubated with KBU2046, and resultant thermolysin reaction products were detected by silver stain following SDS-PAGE. The mean value (from N = 3 independent experiments) of protein bands indicated by arrows is displayed below each lane, and are expressed as the percentage of untreated control. ANOVA P values for changes in band intensity with concentration are displayed. b In silico model of CDC37 (purple) and HSP90β (gray) depicting KBU2046 hydrogen bonding with Gln119 of HSP90β. c Potential surface of the computed ligand binding pocket of the CDC37/HSP90β model with KBU2046 bound. Atoms within 5 angstroms of KBU2046 are colored by element (carbon, green; nitrogen, blue; oxygen, red). d Potential surface of the whole CDC37/HSP90β dimer (color code: green—HSP90β; gray—CDC37; cyan—Arg167 from CDC37 bisecting the larger pocket and creating a new cleft into which KBU2046 binds) Full size image

These combined experiments indicate that KBU2046 only binds to HSP90β and CDC37 when both proteins are present, does not bind to either protein alone, and together support the hypothesis that KBU2046 is binding in a cleft that is only present when CDC37 and HSP90β interact. A comprehensive analysis of HSP90β and CDC37 experimental structural information, including X-ray crystallographic data (PDB IDs: 1uym, 3nmq, 3pry, 2cg9, and 1us7) and chemical cross-linker physical mapping analysis19, supports the notion that CDC37/HSP90β heterocomplex formation results in the formation of a new pocket, that is located at the interface of the two proteins. These modeling studies also predict that KBU2046 binds without any high-energy steric interactions, and with a favorable energy score (Fig. 5b–d and Supplementary Fig. 16). In this computational arrangement, Arg167 from CDC37 protrudes into a large cleft, engages in a hydrogen bond with the carboxyl side chain of Glu33 from HSP90β, which promotes the formation of a new pocket, into which KBU2046 binds.

Together, these findings suggest that KBU2046 binds the CDC37/HSP90β heterocomplex. To examine whether this is associated with an altered signature of bound client kinase proteins, we performed a modified LUMIER assay16 to detect KBU2046-induced changes in client protein binding to CDC37/HSP90β heterocomplexes in intact cells. Of 420 kinase proteins screened, KBU2046 had highly selective effects, significantly changing the binding of only 17 (4%): binding was increased in 10 and decreased in 7 (Fig. 6a and Supplementary Fig. 17a). These findings are in contrast to classical inhibitors of HSP90 function, which have been shown, through this same assay, to affect the binding of the majority client kinase proteins16. Given that TGFβ increases cell motility and that KBU2046 efficacy remains in the face of TGFβ treatment (Supplementary Fig. 11), we repeated the LUMIER assay in TGFβ-treated cells, identifying 3 kinases whose binding to complexes was significantly affected by KBU2046: RAF1 (decreased binding), RIPKI (decreased), and SGK3 (increased) (Fig. 6b and Supplementary Fig. 17a). All three proteins have been shown by others to regulate cell motility, and we demonstrate that knockdown of any one of them decreases motility (Fig. 6c, d and Supplementary Fig. 17b–d). However, only knockdown of RAF1 or of RIPK1 (i.e., the two kinases whose binding to the heterocomplex was decreased by KBU2046) mitigated KBU2046 efficacy, while KBU2046 still retained efficacy in the face of SGK3 knockdown.

Fig. 6 KBU2046-mediated changes in the signature of client proteins bound to the HSP90β/CDC37 heterocomplex mediate effects upon cell motility. a LUMIER assay. HEK293T cells were transfected with 1 of 420 different protein kinases, treated with 10 µM KBU2046 (N = 5 replicates) or vehicle control (N = 5) for 3 days, and LUMIER assays performed. The N = 17 kinases that gave significant findings (Student’s t-test <0.05) in the same direction in each of two separate experiments are depicted. Each separate treatment and kinase condition in each of two separate experiments was conducted in replicates of N = 5. b The experiment was then repeated for these 17 kinases in the presence of TGFβ treatment, and those demonstrating significant differences (t-test <0.05) in the same direction as in a are denoted by *. c, d Wound healing assay. PC3 cells were transfected with siRNA targeting RAF1 (si-Raf1) or non-targeting siRNA (si-control), treated with KBU2046 or vehicle as above, and RAF1 protein measured by western blot (c) and effects upon wound healing measured (d). e Inhibition of RAF1. Purified recombinant HSP90β, CDC37, and RAF1 were combined with KBU2046, as indicated, incubated in an in vitro kinase assay for the indicated times, and western blot for RAF1-Ser338 phosphorylation performed. f, g Effect on HSP90β/CDC37 heterocomplex formation and function in vitro. Purified recombinant HSP90β, CDC37, RAF1, SGK3, or MAP3K6 were combined and treated with KBU2046 or vehicle control, as indicated, incubated in an in vitro kinase assay for the indicated times, and western blot performed, as denoted. All experiments were repeated at separate times at least once, with similar results Full size image

DARTS assay findings (Fig. 5a) supported the notion of a direct interaction. However, the LUMIER-based approach used intact cells treated for 3 days and was unable to determine whether KBU2046 was directly interacting with heterocomplexes. Additional studies were therefore undertaken. Studies focused on RAF1. RAF1 is known to regulate cell motility and metastasis in several cancer types20, while KBU2046’s effect upon RIPK1-complex binding was minor and not further enhanced by TGFβ. KBU2046 did not alter RAF1 protein expression levels in cells (Fig. 6c). This is significant in that HSP90 inhibitors broadly inhibit chaperone activity, thereby decreasing client protein expression. We next constructed an in vitro kinase assay of purified recombinant RAF1, HSP90β, and CDC37, and demonstrated that KBU2046 decreased phosphorylation of RAF1’s Ser338 activation motif (Fig. 6e). In the absence of CDC37/HSP90β heterocomplex, RAF1 activity was much lower, indicating that this effect is heterocomplex-dependent (Supplementary Fig. 18).

As KBU2046 does not directly inhibit protein kinase activity, we hypothesized that its ability to decrease HSP90β phosphorylation resulted from changes in the signature of bound client kinases to the heterocomplex. We examined this by considering that in intact cells KBU2046 increased SGK3 binding to the heterocomplex (Fig. 6a), an effect we anticipated may in turn phosphorylate HSP90β. Utilizing our in vitro kinase assay, we demonstrated that SGK3 increased phosphorylation of HSP90β, and that phosphorylation was further increased in the presence of KBU2046 (Fig. 6f). Recognizing the complexity of the system and the dynamic nature of the client-chaperone complex, we suspected that KBU2046-mediated inhibition of HSP90β phosphorylation was not an isolated event (i.e., not mediated by a single kinase operating in isolation). We explored this possibility in our in vitro system by investigating the interplay of multiple kinases. In intact cells, KBU2046 increases MAP3K6 binding to complexes (Fig. 6a), but MAP3K6 is not predicted to phosphorylate the Ser226 motif. We went on to demonstrate that when MAP3K6 is added to the in vitro kinase assay system, SGK3-mediated phosphorylation of HSP90β is not only inhibited, but in fact decreases in the presence of KBU2046 (Fig. 6g), emulating what is seen in intact cells.