Structure-kinetic relationship reveals the mechanism of selectivity of FAK inhibitors over PYK2

•A helical DFG motif causes slow ligand dissociation rates in FAK but not PYK2•NanoBRET represents a versatile method measuring cells•?RAMD simulation provides tool for accurate ligand-binding kinetic predictions There is increasing evidence of significant correlation between prolonged drug-target residence time and increased drug efficacy. Here, we report structural rationale selectivity two closely related kinases: focal adhesion kinase (FAK) proline-rich tyrosine 2 (PYK2). We found that slowly dissociating inhibitors induce structure at the PYK2. Binding data, high-resolution structures mutagenesis data support role hydrophobic interactions with DFG-helical region, providing from over Our experimental correlate well computed relative times molecular simulations, supporting feasible strategy rationally optimizing times. suggest interplay protein mobility ligand-induced effects key regulator versus Focal (PYK2) are non-receptor kinases constitute subfamily (Schlaepfer et al., 1999Schlaepfer D.D. Hauck C.R. Sieg D.J. Signaling through kinase.Prog. Biophys. Mol. Biol. 1999; 71: 435-478Crossref PubMed Scopus (1019) Google Scholar). Both involved regulation cell migration, adhesion, survival response to extracellular signals, they share similar multidomain organization comprising an N-terminal FERM domain, central catalytic domain (with 61% sequence identity), C-terminal targeting (Lipinski Loftus, 2010Lipinski C.A. Loftus J.C. Targeting Pyk2 therapeutic intervention.Expert Opin. Ther. Targets. 2010; 14: 95-108Crossref (79) Despite their homology, signaling events leading activation differ, being primarily activated by integrins, growth factor receptors, cytokine while PYK2 dependent on intracellular calcium mobilization (Avraham 2000Avraham H. Park S.-Y. Schinkmann K. Avraham S. RAFTK/Pyk2-mediated cellular signalling.Cell Signal. 2000; 12: 123-133Crossref (406) FAKs promote tumor proliferation metastasis overexpressed upregulated several types cancers (Fan Guan, 2011Fan Guan J.-L. Compensatory function promotion (FAK)-null mammary cancer stem tumorigenicity metastatic activity.J. Chem. 2011; 286: 18573-18582Abstract Full Text PDF (46) Scholar; Tai 2015Tai Y.-L. Chen L.-C. Shen T.-L. Emerging roles cancer.Biomed. Res. Int. 2015; 2015: 690690Crossref (154) Wendt 2013Wendt M.K. Schiemann B.J. Parvani J.G. Lee Y.-H. Kang Y. W.P. TGF-? stimulates expression as part epithelial-mesenchymal transition program required outgrowth breast cancer.Oncogene. 2013; 32: 2005-2015Crossref (54) has been be when activity suppressed, suggesting compensatory pathways may result resistance. Efforts have therefore made develop dual FAK/PYK2 currently clinical testing (Lv 2018Lv P.C. Jiang A.Q. Zhang W.M. Zhu H.L. cancer, patent review.Expert Pat. 2018; 28: 139-145Crossref (58) An interesting series aminopyrimidines was reported Pfizer, resulting development PF-562271 (1) (Roberts 2008Roberts W.G. Ung E. Whalen P. Cooper B. Hulford C. Autry Richter D. Emerson Lin J. Kath al.Antitumor pharmacology selective inhibitor, PF-562,271.Cancer 2008; 68: 1935-1944Crossref (318) Scholar), PF-431396 (Buckbinder 2007Buckbinder L. Crawford D.T. Qi Ke H.Z. Olson L.M. Long K.R. Bonnette Baumann A.P. Hambor J.E. Grasser W.A. Proline-rich regulates osteoprogenitor cells bone formation, offers anabolic treatment approach osteoporosis.Proc. Natl. Acad. Sci. U S A. 2007; 104: 10619-10624Crossref (120) defactinib (PF-8554878 [Jones 2011Jones Shapiro G. Bendell Bedard Cleary Pandya Pierce Houk Hosea N. Phase I study PF-04554878, second-generation patients advanced solid tumors.J. Clin. Oncol. 29: 3002Crossref Scholar]), (PF-573228 [Slack-Davis 2007Slack-Davis J.K. Martin K.H. Tilghman R.W. Iwanicki M. E.J. Luzzio M.J. Roberts al.Cellular characterization novel inhibitor.J. 282: 14845-14852Abstract (383) Scholar]) (PF-719 [Tse 2012Tse K.W.K. K.B.L. Dang-Lawson Guzman-Perez Aspnes G.E. Buckbinder Gold M.R. Small molecule modulate chemoattractant-induced Akt follicular marginal zone B cells.Cell. Immunol. 2012; 275: 47-54Crossref (21) inhibitors, respectively. high similarity domains (Figure S1), 1 adopts different back-pocket binding modes proteins 1A). Strikingly, conserved tripeptide DFG-motif forms ?-helical enabling multiple contacts inhibitor kinase, notably pyridine ring L567 1B). In PYK2, same amino acid residues positioned linear conformation only PYK2-D567 able form 1C). This invoked possible explanation approximately 9-fold greater potency (IC50 = 1.5 nM) 13 Recently, studies suggested properties drug, specifically (? 1/koff), provide better indication duration pharmacodynamic action vivo than affinity alone (Copeland, 2016Copeland R.A. The model: 10-year retrospective.Nat. Rev. Drug Discov. 2016; 15: 87Crossref (378) Schuetz 2017Schuetz D.A. Witte W.E.A.d. Wong Y.C. Knasmueller Kokh D.B. Sadiq S.K. Bosma R. Nederpelt I. Heitman L.H. Kinetics Discovery: industry-driven effort target time.Drug Today. 2017; 22: 896-911Crossref (115) observation suggests it mechanism 1. structurally very (10, phenylsulfonamide instead aminopyridine 1) shows (FAK IC50 nM 11 As 10 also show fast kinetics (kon > 106 M?1 s?1, koff 10?2 s?1) (Han 2009Han Mistry Chang J.S. Cunningham Griffor Wang Chrunyk B.A. Walker D.P. al.Structural reveals unique (DFG-out) enables design.J. 2009; 284: 13193-13201Abstract (75) hypothesize about 7-fold FAK—and Scholar) 1—may FAK. often difficult achieve kinases, understanding mechanisms would beneficial future medicinal chemistry efforts. Therefore, this explored features kinetically or balanced FAK/PYK inhibitors. To gain mechanistic distinct profiles binding, performed surface plasmon resonance (SPR) assays, experiments cells, site-directed studies, conventional dynamics (MD) ? random acceleration (?RAMD) simulations. combination techniques allowed us relate dynamic differences Based comparison crystal complex 1), designed synthesized probe influence parts ATP-binding site aminopyrimidine hinge scaffold modified three positions, probing solvent-exposed pocket non-conserved upper lobe (R1), helix induced after (R2), interface (R3) (Table 1). For first assessment effect modifications inhibitor-binding affinity, SPR measurements.Table 1Design structure-activity relationship PYK2FAK (SPR)PYK2 (SPR)Compound no.nameR1R2R3kon (105 s?1)koff (10?2 s?1)KD (nM)kon (nM)1PF-562271-CF353±20.00.34±0.0780.76 ± 0.35130 437.4 2.55.7 1.02-H8.8 2.429 8.8320 279.0 3.375 32820 963-CF30.87 0.220.62 0.2470 220.37 0.151.7 0.78470 1004-CF3n.d.n.d.n.d.n.d.n.d.n.d.5PF-573228-CF370.2 360.82 0.221.3 0,3213 4.833 11250 196-CF317 106.1 4.237 7.017 6.619 7.8110 4.07-CF31.4 0.4414 3.01,000 23no binding8-CF348 207.1 3.015 2.613 8.425 11230 929-CF31.8 0.514.8 1.3260 21no binding10PF-431396-CF330.8 140.054 0.0270.18 0.0558 154.0 1.26.9 0.5411PF-8554878, defactinib-CF324 5.80.44 0.0611.9 0.5015 3.83.6 1.124 3.112PF-719-CF314 6.520 8.3150 1922 8.15.5 2.425 3.3Three substituent sites pyrimidine core 1, R1 (solvent exposed), R2 (DFG adjacent), R3 (back pocket) were considered sites. SPR, resonance; kon, association rate constant; koff, KD, equilibrium n.d., determined. Data n 5 individual experiments. Open table new tab Three these particularly interested interaction located (residues D564–G566), which FAK-L567 into site. pyridyl-methanesulfonamide replaced diverse aromatic moieties (compounds 5–12), modulating electron density pendant system substitution patterns ring. Another polar contact oxindole FAK-R426. conservation residue (R429), observed kinase. contribution difference kinetics, introduced single systems moiety including phenyl (3), ring-expanded 3,4 dihydroquinoline-2-one (4 5), N-methylbenzamide (11, defactinib), N-methylbenzenesulfonamide (12). While did obtain 4, comparable introducing methylsulfonylbenzene (5). position R3, -CF3 group CF3-deleted variant (-H, 2) tested. removal trifluoromethyl resulted reduction compared (1), associated shorter expected back pocket. kept all other investigated. Modifications selected set significantly modulated Interestingly, 4-fold improvement parent compound hypothesized benzene provided more favorable Indeed, if polarity incorporation nitrogen atoms exemplified 11, decreased further. corresponding change off-rate free energy, on-rates 10, similar. affinities parameters agreement lack PYK2-L570. introduction sulfonylphenyl (5) modification accompanied 2-fold increase off-rate. Compared sulfonamide 8 led decrease interestingly, (9) para-fluorobenzene (7) tolerated loss 350-fold 1,300-fold, Introduction indoline-2-one was, however, (6), maintaining low nanomolar affinity. Similar substitutions played determining both profile. Changing (3) 93-fold 60-fold drop on-rate, most likely because does allow formation hydrogen bond Inhibitor 12 PYK2-selective (Tse indeed measured tighter 6-fold against Comparing intriguing kon-koff plot S3) clearly demonstrated had constants kon. Remarkably, variation less 10-fold result, mainly due constant koff. despite values, showed 138 min, orders magnitude slower understand giving rise series, solved six derivatives, R1, R2, 2). slowest off-rate—10—in confirmed overall mode previously described formed bonds backbone C502 region. oxygen atom amino-oxindole (R2) within hydrogen-bonding distance side chain R426. bound cavity created chains M499, D564, L567. (R1) Finally, stabilized amide FAK-D564. discussed here TAE226 S2) (Lietha Eck, 2008Lietha Eck Crystal bis-anilino reveal conformation.PLoS One. 3: e3800Crossref (74) addition, determined 3 5, harboring R1. Intriguingly, revealed loop 4 assumed 180° rotated T-shaped ?-stacking based phenylsulfodioxide without additional present additionally L553. absence methylsulfonamide prevented helix, shown interacted R426 via bond. Changes lead groups. However, R426, appeared transient asymmetric unit. makes involvement modulation inhibitors' on-rate likely. successful solving pointed toward Analysis orientation case direct motif. Hence, its canonical DFG-in arrangement. 3–11. explained impact kinetics. structure-kinetic analysis, FAK, those particular, Simulations potent experimentally All contained sulfoxide coupled results some general trends characteristics rearrangement Compounds higher compounds type-I mode. our published (PDB: 5tob) 3fzr, Figure 3A), 3h3c, 3B) identify crucial relevant either superimposed amino-methyl-phenyl-sulfonamide contrast missing characterized DFG-out preceding P (L431, G432), conformations contribute paralog (Heinrich 2013Heinrich T. Seenisamy Emmanuvel Kulkarni S.S. Bomke Rohdich F. Greiner Esdar Krier Gradler U. al.Fragment-based discovery highly substituted 1H-pyrrolo[2,3-b]- 3H-imidazolo[4,5-b]-pyridines inhibitors.J. Med. 56: 1160-1170Crossref (50) ?C. Although region directly interact one particular P-loop F435 Q432 might affect stabilization G566. FAK-R426 arginine R429 distance. shared scaffold, harbored insights selectivity. Exchanging residues, shift sulfonamide, ?1 kinases. leucine respective L570 stabilizing conformation. secondary 12, seen idea arrangement investigate origin considered, MD carried out simulations ?RAMD (Kokh 2018Kokh Amaral Grädler Musil Buchstaller H.-P. Dreyer Frech Lowinski Vallee al.Estimation ?-random simulations.J. Theor. Comput. 3859-3869Crossref (94) complexes listed Table (omitting 7 9 detected SPR). Conventional protein-ligand ligands line (in ?1, ?C, regions) generally 4A). mobile simulated starting PDB: 5tob, where phenylalanine fragment side-chain variable (for details see S4). explore reasons stability respect fingerprints trajectories. pattern trajectories (Figures 4B 4C), there differences. First, exception 2, D564 methane oriented (see 4D). number larger These included (D564/D567, L569/570), having V484 deep L553 addition V436/V439 A452/A455 Additionally, L501 temporary ligands. packing water-mediated D567 moiety, indicates relatively open partially hydrophilic. 6, DFG-loop observe stable bonding ring, transiently 4B). Next, employed procedure simulate unbinding evaluate studied. (R2 0.91 whole comp