Chemoproteomic selectivity profiling of PIKK and PI3K kinase inhibitors

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Chemoproteomic selectivity profiling of PIKK and PI3K kinase inhibitors Maria Reinecke, Benjamin Ruprecht, Sandra Poser, Svenja Wiechmann, Mathias Wilhelm, Stephanie Heinzlmeir, Bernhard Kuster, and Guillaume Médard ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b01020 • Publication Date (Web): 22 Mar 2019 Downloaded from http://pubs.acs.org on March 23, 2019

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ACS Chemical Biology

Chemoproteomic selectivity profiling of PIKK and PI3K kinase inhibitors

Maria Reinecke1,2,3*, Benjamin Ruprecht1,4*+, Sandra Poser1, Svenja Wiechmann1,2,3, Mathias Wilhelm1, Stephanie Heinzlmeir1, Bernhard Kuster1,2,3,4,5# and Guillaume Médard1#

1 Chair

of Proteomics and Bioanalytics, Technical University of Munich, Freising, Germany

2 German

Cancer Consortium (DKTK), Munich, Germany

3 German

Cancer Research Center (DKFZ), Heidelberg, Germany

4 Center

for Integrated Protein Science Munich (CIPSM), Freising, Germany

5 Bavarian

Center for Biomolecular Mass Spectrometry (BayBioMS), Technical University of Munich, Freising, Germany * These authors contributed equally + Current

address: Chemical Biology, Merck Research Laboratories, Boston, MA, USA

# Correspondence to: Guillaume Médard, [email protected] Bernhard Kuster, [email protected]

ACS Paragon Plus Environment

ACS Chemical Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Chemical proteomic approaches utilizing immobilized, broad-selective kinase inhibitors (Kinobeads) have proven valuable for the elucidation of a compound’s target profile under closeto-physiological conditions and often revealing potentially synergistic or toxic off-targets. Current Kinobeads enrich more than 300 native protein kinases from cell lines or tissues but do not systematically cover phosphatidylinositol 3-kinases (PI3Ks) and phosphatidylinositol 3-kinaserelated kinases (PIKKs). Some PIKKs and PI3Ks show aberrant activation in many human diseases and are indeed validated drug targets. Here, we report the development of a novel version of Kinobeads that extends kinome coverage to these proteins. This is achieved by inclusion of two affinity probes derived from the clinical PI3K/MTOR inhibitors Omipalisib and BGT226. We demonstrate the utility of the new affinity matrix by the profiling of 13 clinical and pre-clinical PIKK/PI3K inhibitors. The large discrepancies between the PI3K affinity values obtained and reported results from recombinant assays led us to perform a phosphoproteomic experiment showing that the chemoproteomic assay is the better approximation of PI3K inhibitor action in-cellulo. The results further show that NVP-BEZ235 is not a PI3K inhibitor. Surprisingly, the designated ATM inhibitor CP466722 was found to bind strongly to ALK2, identifying a new chemotype for drug discovery to treat fibrodysplasia ossificans progressiva.


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Introduction In addition to the typical eukaryotic protein kinases (ePKs), the human kinome also comprises the protein-kinase like group (PKL), whose members share the same PKL-fold and catalytic mechanism as the ePKs but cannot be classified within one of the eight classical ePK groups1, 2. Within the PKLs, the phosphatidylinositol 3-kinases (PI3Ks) and the phosphatidylinositol 3kinase-related kinases (PIKKs) are two structurally related kinase families which share a common origin3. The six human PIKKs are serine/threonine kinases with diverse biological functions2. The family includes the mechanistic target of rapamycin (MTOR) which controls a variety of pathways involved in cell growth and metabolism in response to nutrient and amino acids4, 5. Further members are the gene mutated in ataxia-telangiectasia (ATM) protein and the ATM Rad3-related (ATR) protein which are key switches in DNA damage response6, 7. In contrast, PI3Ks are phospholipid kinases that translate signals from cytokines, growth factors and other stimuli into intracellular signals by phosphorylating the 3-hydroxyl group of phosphatidylinositol derivatives that, in turn, regulate multiple signaling pathways8, 9. PIKKs and PI3Ks represent an important class of drug targets and have been frequently addressed by diverse small molecule kinase inhibitors10,

11.

More than 40 compounds targeting the PI3K-AKT-MTOR pathway have been

tested in clinical trials so far12. To fully understand the modes of action by which such drugs exert their desired or undesired effects, it is important to delineate their range of molecular target proteins. In turn, this knowledge can rationalize or predict failure of clinical candidates due to toxic side effects or serve to repurpose a drug for a new indication13, 14. We and others have shown how chemoproteomics approaches utilizing immobilized, broadly selective kinase inhibitors (Kinobeads) are an efficient and quantitative means to elucidate an inhibitor’s on-target binding and to uncover potential off-targets under close-to-physiological conditions14-17. The Kinobeads technology is a quantitative binding assay that relies on an affinity matrix able to compete with a molecule of interests for binding to target proteins in lysates of cells or tissues15,

16.

In this

chemoproteomic approach, the “target panel” that can be profiled is hence defined by the native proteins that the matrix can specifically bind. We have shown how chemical probes can be derived from known inhibitors to target branches of the human kinome phylogenetic tree (e.g. VEGFRs18, FGFRs19, AKTs20, JAKs21) to cover a larger part of the kinome15, 16. Although, the latest version of Kinobeads captures more than 300 protein kinases from native lysates, the PIKK and PI3K families remained largely uncovered and therefore inaccessible to chemoproteomic selectivity profiling. To extend the Kinobeads assay to those clinically important proteins, we evaluated PIKK and PI3K affinity probes based on the clinical PI3K/MTOR inhibitors Omipalisib (GSK2126458)22



and BGT22623. Bolstered by the addition of these two probes, and without sacrificing ePKs coverage, we used the new version of Kinobeads (termed Kinobeads ε) to generate selectivity profiles for 13 pre-clinical and clinically evaluated PI3K and PIKK inhibitors. We show that results from Kinobeads ε are a better approximation of compound action of PI3K inhibitors in-cellulo than recombinant kinase assays and we identified ACVR1 (ALK2) as a novel potently-bound and inhibited target of the ATM inhibitor CP46672224 that may offer new opportunities for the use of this compound.


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Results and Discussion Immobilized BGT226 and Omipalisib analogue can enrich PI3Ks and PIKKs We screened the literature for potent small molecule ATP-competitive PIKK and PI3K inhibitors that could be immobilized onto a solid matrix. Among others, the commercially available dual PI3K/MTOR inhibitor BGT22623,

25

with IC50 values of 4 nM, 63 nM and 38 nM for PIK3CA,

PIK3CB and PIK3CG (PI3Kα/β/ɣ) respectively could be directly coupled to NHS-activated beads owing to the presence of a piperazine amine (Figure 1A). BGT226 shares the tricyclic imidazoquinolinone ring core of NVP-BEZ235 (Dactolisib; Figure 1A) and it has been reported that NVP-BEZ235 potently inhibits PIK3CA, PIK3CB, PIK3CG, MTOR and ATR26, 27. Omipalisib cannot be directly coupled to beads but its widespread use and extraordinarily potent affinity for all PI3K isoforms motivated us to design a linkable analogue of Omipalisib (Figure 1B)22. We found

the

pyridazine-replacing

benzoic

acid

analogue

CAS1313994-59-0

(Patent

WO201108228528) to be the analogue of choice because it can be immobilized by an amide bond. We were comforted in this decision on the basis of the reported activity of the pyrrolidine ester analogue (CAS1607009-17-5; IC50 of 0.70 nM vs 0.77 nM for Omipalisib against PIK3CA in the Kinase-Glo® assay; Figure 1B; Patent WO201406747329) and the X-ray structure of the co-crystal of Omipalisib with PIK3CG (PDB: 3L0822) indicating that the carboxylic acid should be pointing towards the solvent (Figure 1C). We therefore synthesised this molecule following the route reported for the Omipalisib series (Supporting Information and Supporting Scheme S1)22, that we have already used in a classical target deconvolution experiment of Omipalisib14. We functionalized NHS-activated Sepharose beads with BGT226 and the linkable analogue of Omipalisib (referred to as iBGT226 and iOmipalisib from here on) and performed affinity pulldown experiments using a cell lysate mixture of four cancer cell lines (MV-4-11, K-562, COLO-205 and SK-N-BE(2)) followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) readout, protein identification and quantification as described14. The affinity matrices efficiently captured PIKKs and PI3Ks and specificity of the enrichment was confirmed by competition experiments using the free respective inhibitor (Figure S1; Supporting Information). More specifically, iBGT226 enriched PIK3C2A, PIK3C2B, PIK3CA, PIK3CG, PIK3C3, MTOR, PRKDC, ATM, and ATR (Supporting Table S1A) and iOmipalisib showed specific enrichment of PIK3C2A, PIK3C2B, PIK3CA, PIK3CB, PIK3C3, PI4KA, PI4KB, PRKDC and MTOR (Supporting Table S1B). Addition of new affinity probes extends the coverage of Kinobeads to PI3Ks and PIKKs



We next sought to combine iBGT226 and iOmipalisib with the established version of Kinobeads (KBγ)16 so that binding affinities of a compound towards PIKKs, PI3Ks and the ePKs captured by KBγ may be determined within one experiment. First, we combined iOmipalisib beads with the KBγ matrix in different mixing ratios: iOmipalisib:KBγ in a ratio of 1:1, 1:5 or KBγ alone, and performed a 10 dose competitive pulldown experiment (0.3 nM - 1 µM including pulldown of pulldown experiment to correct for target depletion and calculate apparent Kdapp values16) using Omipalisib as competitor and lysate mixture of MV-4-11, SK-N-BE(2), COLO-205, K-562 and OVCAR-8 cells. In the resulting dose-response curves, we found that some PIKK family members such as MTOR and PRKDC could not be completely competed when the proportion of iOmipalisib in the bead mixture was too low (1:5 ratio, Figure 2A, Supporting Table S2A). We suspect those proteins to be allosterically bound by one of the KBγ affinity probes preventing competition by the ATP-pocket binders. We also noted that the number of captured ePKs did not significantly diminish with a higher ratio of iOmipalisib (1:1) while the number of quantified PIKKs, PI3Ks and PI4Ks remained identical (Figure 2B). This led us to choose a KBγ:PIK(K)-matrix ratio of 1:1 to maximize the dynamic range of the observable competition. Next, we examined the identity and intensity of the proteins captured by KBγ in combination with our novel matrices more closely. Specifically, we created three bead mixtures: i) KBγ supplemented with iBGT226, ii) KBγ supplemented by iOmipalisib and iii) KBγ supplemented by both iOmipalisib and iBGT226 (termed KBε) and compared these against KBγ. We performed triplicate pulldown experiments using the aforementioned five cancer cell line lysate mixture as described16. The results showed that nearly all PIKKs and PI3Ks (except for TRAPP) were statistically significantly enriched (p-value < 0.01) by KBε over KBγ (Figure 2C; S2A-C; Supporting Table S2B). ATM and ATR were mainly enriched by iBGT226 whereas iOmipalisib led to a stronger enrichment of PI3Ks (e.g. PIK3CA and PIK3CB, Figures S2C). Of note, we also observed interaction partners of several PIKK and PI3K family members, including MLST8 which is a known interactor of MTOR or PIK3R1. In addition, the KBε enriched several metabolic enzymes (ACOX1, ALDH9A1, NQO1 or CPOX, Figure 2C). If this enrichment were specific, kinobeads may provide an assay for those non-kinase proteins in the future. We have already reported such unexpected off-targets of kinase drugs including NQO2 and FECH that are commonly engaged by small molecule kinase inhibitors14, 30. For example, like NQO2, ACOX1 has FAD as cofactor and CPOX has a binding site for heme such as FECH, which renders these proteins highly probable to bind specifically to KBε.


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Last, we used the very unselective protein kinase inhibitor AT-9283 to compare target Kdapp values between KBε and KBγ14 (Figure S2D; Supporting Table S2C). We chose AT-9283, because its more than 100 targets cover a broad spectrum of proteins (different protein kinase families, nonkinase targets) across an affinity range of four orders of magnitude. The target affinities determined by the two affinity matrices were reasonably well conserved (correlation of R=0.73). The discrepancies can be explained by a few outliers caused by technical and/or biological variation. For instance, EPHA7 and MET were represented by few unique peptides in only one of the two datasets (see Supporting Table S2C). The new affinity matrix, KBε, was as good for ePKs as KBγ but also allowed studying drug interactions for 14 out of 17 PIK and PIKK kinases (Figure 2D). Selectivity profiling of 13 PI3K and PIKK inhibitors using KBε To demonstrate the applicability of KBε, we profiled two ATM, two ATR, one PIK3CA and 8 dual MTOR/PI3K inhibitors using a competition pulldown workflow (Supporting Table S3A; all drugtarget profiles are provided as PDF files on ProteomeXchange and ProteomicsDB31)16. Here, compounds were dosed at nine concentrations ranging from 1 nM to 10 µM plus vehicle and target depletion control in a combined cell lysate of five cancer cell lines (MV-4-11, SK-N-BE(2), COLO-205, K-562 and OVCAR-8) (Figure S1). The pKdapp values (negative logarithm of apparent dissociation constants Kdapp in molar) for all 13 kinase inhibitors and their corresponding targets were assembled in a drug/target interaction matrix using unsupervised clustering (Figure 3, Supporting Tables S3B, S3C, S3D). As expected, the PIKK and PIK protein families were predominantly targeted by the inhibitors with high binding affinities (PIKK, PI3K, PI4K are marked in pink; pKdapp values mostly greater than 7). Omipalisib stood out as a pan-PI3K and PIKK inhibitor with very high affinities (pKdapp values above 7). Beside the members of these two families, no other proteins were found to interact with Omipalisib. BGT226 was identified as a pan-PIKK inhibitor binding to MTOR, PRKDC, ATM and ATR with very high affinities (Kdapp of 3 nM, 8 nM, 0.6 nM and 4 nM), whereas the PI3K and PI4K family was targeted with lower potency (pKdapp values around 7), except for PIK3CA. The six additional pan MTOR/PI3K inhibitors Apitolisib, Gedatolisib, NVP-BEZ235, PF-04691502, VS5584 and Voxtalisib also bound to several members of the PIKK and PI3K families but in general with much weaker affinity compared to Omipalisib and BGT226 (pKdapp values around 6 to 7) validating a posteriori our initial probe design. For each of the PI3K and PIKK proteins that were identified as targets of at least one of the tested compounds, we ranked the 13 molecules according to their selectivity using the concentration



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and target dependent selectivity (CATDS14) scoring scheme (Supporting Table S3E, for details see Supporting Information and

14).

CATDS scales from 0 (low selectivity) to 1 (high selectivity)

and provides a more sophisticated measure of selectivity than e. g. counting the number of targets at a fixed concentration. Using CATDS, we identified Voxtalisib as the most selective PIK3CG inhibitor within our screen (Figure S3A; Supporting Table S3E). Even if higher doses of Voxtalisib led to inhibition of 12 further targets (including several PI3K and PIKK members, ACAD11, NQO2 and casein kinases, Figure S3B, Supporting Table S3D), a CATDS score of 0.78 at an inhibitor concentration of 14 nM shows that the drug is first and foremost a PIK3CG inhibitor, in line with previous work32, 33. Literature in vitro kinase inhibitor assay data for Voxtalisib showed IC50 values of 39, 110, 43, 9 and 150 nM for PIK3CA, PIK3CB, PIK3CD, PIK3CG and PRKDC33. We found Kdapp values of 69 nM, 1.3 µM, 14 nM and 228 nM for PIK3CA, PIK3CD, PIK3CG and PRKDC respectively in our binding assay (Supporting Table S3G). In addition, the previous study reported MTORC1 and MTORC2 inhibition at 160 and 190 nM in an immune-complex kinase assay and no other kinase targets among 150 measured kinases except CSNK1D that showed a weak IC50 of 1.55 µM. In our assay, MTOR was not bound by the drug suggesting that it cannot bind to the ATP pocket with good affinity. We did however find CSNK1D and CSNK1E to be bound with Kdapp values of 497 and 248 nM respectively. We further compared the binding affinities for PI3K obtained by the Kinobeads assay to literature data generated using recombinant kinase assays and observed discrepancies, particularly for NVP-BEZ235 (Supporting Table S3G). This compound was reported as a nanomolar dual inhibitor of PI3Ks and MTOR26 with potencies of 4 nM, 75 nM, 5 nM and 7 nM for PIK3CA, PIK3CB, PIK3CG and PIK3CD respectively but our pulldown assays did not show any strong effect (Supporting table S3C). Bergamini and coworkers34 had already shown that NVP-BEZ235 inhibited PIK3CA only at much higher concentrations and did not interact with PIK3CG at all, which was confirmed by a cellular assay in which NVP-BEZ235 failed to inhibit PIK3CG-dependent migration of primary human granulocytes34. Global

phosphoproteomics

shows

that

NVP-BEZ235

does

not

inhibit

the

PI3K/PREX1/MEK/ERK axis in-cellulo The significant differences observed between the data from in vitro kinase panels and affinity matrix pulldowns obtained with KBε raised the question which values are relevant for predicting response in a cellular assay. To address this, we sought to measure proximal cellular PI3K target engagement markers using global quantitative phosphoproteomics. The main advantage of a phosphoproteomics approach over other functional cellular assays is that MTOR and PI3K


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signaling can be distinguished by different substrate proteins that get phosphorylated. We treated BT-474 breast cancer cells which are characterized by high PI3K signaling activity as a result of activating PIK3CA mutations35 outside the kinase domain with 1 µM NVP-BEZ235 or 1 µM Omipalisib for 1 h in four biological replicates and used mass spectrometry based phosphoproteomics to quantify changes in ~15,000 distinct phosphorylation sites. After stringent filtering and statistical analysis (see Supporting Information and Supporting Table S4A), we found that phosphorylation of several known MTOR substrates was almost completely suppressed after treatment with Omipalisib and NVP-BEZ235 (e. g. AKT1S1 pS-203; Figure 4A), indicating that both inhibitors are cell permeable and both bound and inhibited MTOR. According to two recent studies, PI3K controls MAPK1/3 activation in BT-474 cells via a PREX1-RAF signaling axis36, 37. In contrast to MTOR substrates, phosphorylation of MAPK1 (pY187) and MAPK3 (pT202/pY204), all of which are indicators of MAPK1/3 kinase activity was only reduced following treatment with Omipalisib but not with NVP-BEZ235 (Figure 4B and C). Western Blot analysis of MAPK1/3 pT202/pY204 after treating BT-474 cells with different concentrations of NVP-BEZ235 confirmed that phosphorylation of MAPK1/3 was not suppressed by the compound (Figure 4D, Figure S4A). We also observed strong inhibition of PREX1 pS1182 (log2FC = -1.9, p = 6.6E-04) and RAF1 pS29 (log2FC= -1.0, p =4.6E-03) for Omipalisib but not NVP-BEZ235 which further confirmed the sustained activity of PI3K after NVP-BEZ235 treatment (Figure 4B, 4C). Taken together the global phosphorylation analysis showed no PI3K inhibition by NVP-BEZ235 in-cellulo which agrees well with the Kdapp values (Kdapp of 236 nM, 422 nM and >10 µM for PIK3CA, PIK3CD and PIK3CG respectively) obtained from the chemoproteomic experiment. Further, we investigated the effect of NVP-BEZ235 or Omipalisib treatment on the PI3K/AKT/MTOR pathway by Western Blot analysis of AKT phosphorylation status. It is widely accepted that PI3K phosphorylates AKT T308 via PDK1, leading to kinase activation38. Maximal activation is then achieved by phosphorylation of AKT S473 by MTOR complex 2 (MTORC2). Here, we monitored those two key phosphorylation sites of AKT to distinguish between PI3K and MTOR signaling. Accordingly, BT-474 cells were treated with either DMSO or 1 µM of one of the following inhibitors: AZD-8055 (selective MTOR inhibitor), CH5132799 (selective PI3K inhibitor), NVP-BEZ235 or Omipalisib. Unexpectedly, the phosphorylation of AKT T308 was reduced by all four inhibitors in comparison to DMSO: by 97 % after Omipalisib treatment, 89 % after NVP-BEZ235 treatment, 46 % after AZD-8055 treatment and 93 % after CH513799 treatment (Figure 4E and Figure S4B). Additionally, phosphorylation of AKT S473 was totally abolished by all four molecules (Figure 4E, Figure S4C). Because the MTOR-selective

AZD-8055

and

the

PI3K-selective

CH5132799

inhibitors

both

elicit

dephosphorylation of the two activation sites of AKT in BT-474 cells, it becomes uncertain whether



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those sites can be used as markers of PI3K- or MTOR-only drug engagement. A more intricate mechanism seems to be at play where the interdependence of those kinases render the elucidation of their individual action perilous. As a result, it appears that great care has to be taken when interpreting PI3K kinase activity data obtained using recombinant enzymes39, while PI3K drug action in-cellulo is likely not best apprehended by analyzing AKT phosphorylation status. Whereas NVP-BEZ235, according to KBε analysis, efficiently engages MTOR (Kdapp = 31 nM) but not PI3K, we also found high affinities for PRKDC (29 nM), ATM (13 nM) and ATR (8 nM). Interestingly, NVP-BEZ235 was found to be the most selective ATR inhibitor of all 13 compounds tested (CATDSATR of 0.59 at a compound concentration of 7.6 nM). Overall, these results clearly call for a re-definition of the initially proposed mechanism of action for this compound. The ATM inhibitor CP466722 is a potent ALK2 binder Among the two designated ATM inhibitors (CP46672224 and KU6001940) and five additional inhibitors found to target ATM in this screen (Figure 5A; Supporting Table S3E), we found KU60019 to be exceptionally selective for ATM (CATDS score of 1 at a Kdapp of 4 nM for ATM, Figure 5B). In contrast, the designated ATM inhibitor CP466722 (Figure 6A) did not demonstrate selectivity towards ATM (Kdapp of 51 nM, CATDS of 0.14; Figure 6B). But interestingly, amongst the 25 additional targets of CP466722, ACVR1 (ALK2, Kdapp of 33 nM) was found to be the most potently engaged (Figure 6B, Supporting Table S3D). Kinase activity assays further confirmed inhibition of wild-type ACVR1, constitutively active ACVR1(Q207D) and disease-relevant ACVR1(R206H) (IC50 of 729 nM, 458 nM and 887 nM respectively; Supporting Table S5). Within the Kinobeads screen CP466722 was the most selective compound for ACVR1 with a CATDSACVR1 scores of 0.21 at a Kdapp of 33 nM. When comparing CATDSACVR1 scores for 23 additional molecules found to bind ACVR1 in our previously published clinical kinase inhibitor profiling study14, CP466722 was still the most selective (Figure 6C, 6D, Supporting Table S3F). The affinity and selectivity of CP466722 for ACVR1 struck us as particularly interesting owing to the therapeutic potential of this kinase. Activating mutations of ACVR1 have been identified for the two unrelated pediatric diseases fibrodysplasia ossificans progressiva (FOP) and diffuse intrinsic pontine gliomas (DIPG). For DIPG, the role of ACVR1 and its various mutations41,

42

needs further investigation to validate the inhibition of its kinase activity as a therapeutic opportunity43. In the case of FOP however, 97% of patients harbor the activating R206H mutation in the glycine-serine activation domain of the protein, making intervention on ACVR1 a major therapeutic hope44, 45. There is currently no curative treatment for this debilitating orphan disease, where

episodes

of

spontaneous

heterotopic

ossification


lead

to

premature

death.

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Phylogenetically, ACVR1 is a serine threonine kinase receptor (STKR) which belongs to the tyrosine kinase-like group (TKL) of kinases. The STKR family comprises 12 member in humans, named ACVR1 (ALK2); ACVR1B (ALK4); ACVR1C (ALK7); ACVR2A; ACVR2B; ACVRL1 (ALK1); BMPR1A (ALK3); BMPR1B (ALK6); BMPR2; TGFBR1 (ALK5) and the structurally related TGFBR2 and AMHR22. Among these, nine can be captured by KBε but only ACVR1 showed potent competition by CP466722 (Kdapp of 33 nM). We noted that such high intra-family selectivity has not been described for any reported ALK2 inhibitor (including KRC203, KRC360, LDN193189, LDN214117, LDN212838, K0228, Dorsomorphin, LDN212854, DMH1, K02288a46-48 and the novel quinazolinone inhibitors published recently49) which renders this chemotype interesting in the lookout for ACVR1 drugs. Future co-crystallisation of CP466722 with ACVR1 should help decipher the molecular features responsible for the intra-family selectivity. While semi-flexible docking using three co-crystals (Figure S4A) predicted the pyrimidine of CP466722 to establish the hinge binding (Figure S4B), rigid docking of CP466722 in 3Q4U using Molecular Forecaster50 indicated a favored pose, where the triazolamine is the key binding element involved in two hydrogen bonds (Figure S4C). Here, the triazolamine acts as an isostere of the aminopyridine of K02288 (PDB:3MTF) or the pyrazole moiety of LDN193189 (PDB:3Q4U) while the quinazoline moiety mimicks the quinoline of LDN193189. Future chemoproteomic-aided medicinal chemistry efforts can delineate the kinome-wide structure-activity relationships of this chemotype en route to the discovery of ACVR1 drugs51, 52.

Conclusions With this work, we have extended the reach of Kinobeads to the profiling of PIKK and PI3K inhibitors. Endowed with this tool, we profiled a set of 13 PI3K and PIKK inhibitors and classified them according to their selectivity notably for ATM, for which KU60019 was found to be an excellent tool. Surprisingly, CP466722 was identified as a potent ALK2 inhibitor with exquisite intra ALK-family selectivity. This singularity makes this molecule an interesting lead in the search for drugs addressing fibrodysplasia ossificans progressiva. In addition, phosphoproteomic experiments demonstrated that chemoproteomics data better reflect in-cellulo PI3K inhibitor action than recombinant kinase assays. As a result, previously delineated compound mechanisms of action need re-defining. Overall, the extension of the Kinobeads matrix technology to encompass the profiling of PI3K and PIKK inhibitors serves the exploration of cross-reactivities across the kinome. Such efforts are instrumental for both the understanding of drug action and for pharmacophore repurposing.

Methods



Detailed experimental methods are provided in the Supporting Information.

Accession Codes The mass spectrometry proteomics data and drug dose response curves have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE53 partner repository with the dataset identifier PXD011719 (Reviewer account: Username: [email protected] Password: 9vngHuIy). Additionally, drug dose response data will be made available in ProteomicsDB (https://www.proteomicsdb.org/).

Acknowledgments The authors thank S. Sieber and E. Kunold, TUM Department for Chemistry, for measuring the phospho-proteome samples. The authors also thank A. Hubauer, M. Krötz-Fahning, and A. Klaus for technical assistance.

Funding Sources The present study was in part funded by the German Cancer Consortium for Translational Research DKTK and by the Center for Integrated Protein Sciences Munich, CIPSM. The authors declare the following competing financial interest(s): B. Kuster and M. Wilhelm are founders and shareholders of OmicScouts GmbH, Freising. They have no operational role in the company.

Supporting Information Supporting Information available: This Material is available free of charge via the Internet. 

Supporting Figure S1-S4 and Scheme S1, legends for Supporting Tables 1-4 and Data Sets 1 and 2, Materials and Methods



Supporting Excel Table S1 (immobilized BGT226 and Omipalisib analogue)



Supporting Excel Table S2 (new affinity matrix)



Supporting Excel Table S3 (profiling of PIKK and PIK inhibitors)



Supporting Excel Table S4 (phosphoproteomics dataset)



Supporting Excel Table S5 (kinase activity assay)



Supporting Data S1 (CP466722Docking_3q4u)



Supporting Data S2 (CP466722Docking_3q4u_3mtf_6gin)


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Figure Legends Figure 1 Chemical structures of novel PIKKs and PI3Ks affinity probes and their parent compounds. A) Structure of the commercially available pan-PI3K inhibitor BGT226 that shares the tricyclic imidazoquinolinone ring core with the pan MTOR/PI3K and ATR inhibitor NVP-BEZ235. BGT226 can be directly immobilized on Sepharose beads via its piperazine amine to yield iBGT226. B) Chemical structure of Omipalisib and its pyridazine-replacing benzoic acid/ester analogues. The benzoic acid can be immobilized on Sepharose beads via its carboxylic acid moiety to yield iOmipalisib. The pyrolidine ester analogue has been reported to exhibit similar activity on PIK3CA as Omipalisib29. C) Co-crystal structure of PIK3CG and Omipalisib, indicating that the carboxylic acid of the analogue points towards the solvent (PDB: 3L08).

Figure 2 Addition of iOmipalisib and iBGT226 to KBγ allows for PIKK and PI3K inhibitor profiling. A) Dose response curves for MTOR after competitive pulldown experiments with different bead ratios of iOmipalisib and KBγ beads (KBγ:iOmipalisib of 5:1 or 1:1 or KBγ alone) using 10 doses of Omipalisib as free drug competitor (0.3 nM – 1 µM). B) Number of competed PI3Ks, PI4Ks and PIKKs and number of identified ePKs in competitive pulldown experiments. C) Volcano plot comparing proteins captured by KBγ and KBε in a triplicate experiment. The significance of the differences was tested in a two-sided t-test (S0= 0.1, 0.01 FDR). PIKKs, PI3Ks and PI4Ks (labeled in pink) were significantly enriched by KBε. Proteins exhibiting significant differences are colored in grey. D) Part of the kinome tree showing the PIK and PIKK families. Kinases that can be enriched and competed using KBε are marked in pink.

Figure 3 Selectivity profiling of 13 PI3K and PIKK inhibitors Unsupervised hierarchical clustering of proteins competed by the 13 PIKK and PI3K inhibitors tested, (color code indicates the pKdapp bin of the drug-target interaction). PIKKs, PI3Ks and PI4Ks are marked in pink.

Figure 4 Cellular PI3K and MTOR target engagement. A) Residual phosphorylation of several known MTOR substrates after treating BT-474 cells with 1 µM NVP-BEZ235 or 1 µM Omipalisib for 1 h. Error bars depict the standard deviation in four



biological replicates. Phosphorylation was reduced after both drug treatments showing that both inhibitors are cell permeable and inhibit MTOR. B) Residual phosphorylation of different phosphorylation sites involved in the PI3K-MAPK1/3 pathway after treating cells with Omipalisib or NVP-BEZ235. Inhibition of PREX1 pS1182 and RAF1 pS29 was exclusively observed after Omipalisib treatment. Error bars indicate standard deviation of four biological replicates. C) Volcano plot showing log2 fold changes of quantified phosphorylation sites after treating BT-474 breast cancer cells with 1 µM Omipalisib or 1 µM BEZ235 for 1 h. Phosphorylation of MAPK1 pY187 and MAPK3 pT202/pY204 (PI3K downstream signaling) were only reduced after treatment with Omipalisib. Phosphorylation sites exhibiting significant changes (FDR

Chemoproteomic selectivity profiling of PIKK and PI3K kinase inhibitors

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