Reviving B-Factors: Activating ALK Mutations Increase Protein

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Letter

Reviving B-factors: Activating ALK mutations increase protein dynamics of the unphosphorylated kinase Ted W Johnson, Ben Bolanos, Alexei Brooun, Rebecca A. Gallego, Daniel Gehlhaar, Mehran Jalaie, Michele A McTigue, and Sergei L Timofeevski ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00348 • Publication Date (Web): 24 Aug 2018 Downloaded from http://pubs.acs.org on August 24, 2018

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ACS Medicinal Chemistry Letters

Reviving B-factors: Activating ALK mutations increase protein dynamics of the unphosphorylated kinase Ted W. Johnson,* Ben Bolanos, Alexei Brooun, Rebecca A. Gallego, Dan Gehlhaar, Mehran Jalaie, Michele McTigue, Sergei Timofeevski Pfizer Worldwide Research and Development, La Jolla Oncology, 10770 Science Center Drive, San Diego, California 92121, United States KEYWORDS B-factor, normalized, anaplastic lymphoma kinase, ALK, kinase, protein, dynamics, mutation, activation, crystallography, x-ray ABSTRACT: Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that can become oncogenic by activating mutations or overexpression. Full kinetic characterization of both phosphorylated and non-phosphorylated wildtype and mutant ALK kinase domain was done. Our structure based drug design programs directed at ALK allowed us to interrogate whether x-ray crystallography data could be used to support the hypothesis that activation of ALK by mutation occurs due to increased protein dynamics. Crystallographic B-factors were converted to normalized B-factors which allowed analysis of wildtype ALK, ALK-C1156Y and ALK-L1196M. This data suggests that mobility of the P-loop, αC-helix, and activation loop (A-loop) may be important in catalytic activity increases, with or without phosphorylation. Both molecular dynamics (MD) simulations and hydrogen deuterium exchange experimental data corroborated the normalized B-factors data.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase and clinical target in oncology.1 ALK mutation or overexpression has been implicated in a variety of tumor types. For example, ALK-fusion proteins that arise from gene translocations define a distinct molecular subtype of non-small-cell lung cancer (NSCLC) and lead to constitutive activation of the kinase and tumor formation.2 Oncogenic ALK-fusions are most common in NSCLC, but have also been reported in other tumor types.3 Primary single amino acid missense mutations cause ligandindependent activation of the receptor tyrosine kinase ALK and are a major cause of neuroblastoma,4 the most common cancer in children under 12 months of age. Frequent mutations include F1174L, R1275Q, K1062M, Y1238S, L1196M, and T1151M.5 Gain of function mutations in the intact ALK gene have also been reported in anaplastic thyroid cancer.6 Since introduction of the first approved ALK inhibitor, crizotinib,7 reports of secondary missense mutations have been reported and are a major cause of acquired resistance in NSCLC.8 Reported crizotinib acquired resistance mutations include L1196M, G1269A, E1210K, S1206Y/C, G1202R, I1171T/N/S, C1156Y, and 1151Tins.9-11 Following treatment with second-generation inhibitors (ceritinib, alectinib, and brigatinib) reported mutations include G1202R, F1174L/C, L1196M, I1171T/N/S, C1156Y, E1210K, D1203N, G1202del, and V1180L.12 Of the second generation mutations, G1202R is by far the most frequent. In addition, some primary missense mutants in neuroblastoma are not sensitive to crizotinib treatment due to its modest ALK potency. Mechanisms involving ALK function can alter activation rates, catalytic activity and binding of ATP, inhibitor or protein substrate.13 Treating acquired resistance and brain metastases have been a focus of

many groups, leading to second10, 14-16 and third generation17 inhibitors of ALK. To better understand ALK mechanisms of resistance with experimental data, we investigated the protein dynamics with multiple techniques and correlated those changes with catalytic activity of ALK oncogenic mutants. The non-phosphorylated ALK kinase domain (KD) represents the basal kinase state. Overall, neuroblastoma-derived ALK mutations and many ALK-fusion secondary point mutations have their greatest effects on the activity of unphosphorylated ALK KD, promoting its constitutive autophosphorylation and, thus, ligand-independent signaling by the intact receptor.13 As a baseline, auto-phosphorylated, activated states of ALK increase kcat for WT by 143-fold relative to unphosphorylated enzyme (Table 1). The likely mechanism for the ligand, dimer or oligomer-induced activation of ALK involves the trans-phosphorylation of an activation loop (Aloop) tyrosine (Y1278) by the partner ALK protein kinase followed by trans-phosphorylation of the other two A-loop residues (Y1282 and Y1283).18 Several kinetic and binding parameters were calculated for the non-activated and activated constructs of ALK kinase domain. The turnover rate, or kcat (s-1), and Michaelis constants for both ATP and protein substrate for several reported clinical mutations are shown in Table 1. Catalytic efficiencies were calculated relative to WT enzyme. Phosphorylated protein kinetics showed little to no increase in activation. On the contrary, the unphosphorylated proteins show increased catalytic efficiency. For example, ALK-C1156Y and the gatekeeper mutant (L1196M) significantly increased catalytic efficiency of processing ATP using non-phosphorylated ALK by 8- and 11-fold, respectively, and processing phosphoacceptor substrate by 14- and 40-fold, respectively. Based on the kinetic

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parameter results, we focused on unphosphorylated WT ALK KD and the gain of function resistant mutations C1156Y and L1196M, with the same protein used for crystallography experiments. We hypothesized that the increase in catalytic efficiency of the mutants could be due to spatially differentiated increased protein dynamics. Table 1. Non-activated vs. activated ALK KD kinetic parameters ALK

kcat

KM,ATP

KM,YFF

Relative

Relative

variant

(s )

-1

(mM)

(mM)

kcat/KM,ATP

kcat/KM,YFF

WT

0.077

239

823

1.0

1.0

C1156Y

0.44

166

341

8.4

14

L1152R

0.65

108

285

19

24

F1174L

2.2

341

950

20

24

L1196M

0.52

147

136

11

40

pWT

11.0

116

197

1.0

1.0

pC1156Y

39.7

78

165

5.4

4.3

pL1152R

18.1

100

247

1.9

1.3

pF1174L

19.0

74

181

2.7

1.8

pL1196M

22.8

79

79

3.1

5.2

Proteins are flexible and highly dynamic. Protein motion varies from picosecond-scale rapid vibrations to conformational changes that take up to 1,000 seconds and movement greater than 10 Å.19 The dynamic nature of protein structures has been recognized, established, and accepted as an intrinsic fundamental property with major consequences to their function. For example, protein internal motion and flexibility is important for functions such as activation, catalysis and allostery.20 Choy and coworkers studied structural and dynamic data for PTP1B and concluded that catalysis and hydrolysis required both protein rigidity and slow dynamics while its allosteric regulation required fast dynamics.21 There is also growing support for shorter-timescale fluctuations within proteins impacting longer-timescale conformational changes.22-25 Ultimately, controlling the temporal and spatial components of motion are critical for an enzyme to function properly.26 The activation loop (A-loop) of kinases is a key regulator of kinase activity.27 Auto-inhibition using activation loop residues or inhibition by other proteins prohibits substrate binding. For example, the activation loop of insulin receptor kinase collapses into the active site preventing substrate from binding.28 In another example, imatinib binds to the inactive conformation of ABL that mimics bound substrate.29 Activation by stabilization of the active conformation or destabilization of inactive conformations can occur by several mechanisms, a) phosphorylation (typically activation loop residues), b) separate regulatory proteins and domains, c) amino acid mutation, and d) use of insertions or extensions in the kinase domain.30 Each of these mechanisms can be sufficient alone in kinase activation, or in combination. Activating mutants destabilize inactive conformations and mimic the conformational changes promoted by activation, sometimes obviating the need for Aloop phosphorylation, as with V599EBRaf.31 Increased protein kinase dynamics, in particular A-loop dynamics, can destabilize quiescent conformations and open inhibited sites to substrate binding,20 increasing autophosphorylation and trans-

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phosphorylation of substrates in the presence or absence of activation loop phosphorylation. Crystallographic B-factors (B), also called atomic displacement factors or temperature factors, are included for all structures deposited to the protein data bank (wwpdb.org) and are calculated coefficients that compensate for the difference between observed diffraction and the theoretical diffraction of an atom that is not moving. B-factors are correlated with atomic motion and can provide insight into the dynamics of proteinligand complexes.19 To compensate for absolute value differences across crystallographic data, we utilized normalized Bfactors (B') that allowed comparisons across distinct crystal experiments. B' scoring normalizes the B-factors for each atom by subtracting the mean (µB) and dividing by the standard deviation (σB), calculated across all non-hydrogen atoms in each protein structure (Equation 1). This is also known as a Zscore. B' = (B – μB) / σB.

(Equation 1)

Others have used both B-factors and normalized B-factors to link dynamics to enzyme activity. Ho and coworkers used a B-factor analysis to identify important regions of drug binding and transport in AcrB.32 They found that region 3 has enormously large B-factors relating to transporter protein function. In addition, Siglioccolo et al. studied cold-adapted proteins and found higher normalized B-factors than their mesophilic counterparts, suggesting increased dynamics allowing the enzymes to function at lower temperatures.33

Figure 1. Co-crystallized WT ALK (apo) colored by normalized B-factor (-2 to 0 to +2; red to white to blue).

Normalized B-factors were calculated using Equation 1 for structures of un-liganded, non-phosphorylated ALK KD: WT, C1156Y, and L1196M. The crystal forms, and therefore the crystal contacts, are the same for all three structures. Figure 1 highlights WT protein which serves as a baseline. Normalized B-factors for displayed ribbons-rendering are colored by the normalized B-factor of Cα atoms on a per-residue basis. Regions of the protein which could not be modeled due to uninterpretable electron density, indicating high dynamics, include


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ACS Medicinal Chemistry Letters residues 1124−1130 (in the P-loop), residues 1136−1143 (loop connecting β2 and β3), residues 1280−1285 (in the A-loop), and residues 1401−1411 at the C-terminus of the protein construct. Regions of the modeled protein structure that have high normalized B-factors are the N and C-termini, the P-loop, the A-loop, and the C-terminal domain loop (1212−1222) connecting αD and αE.

downstream residues (1151−1165), and αD-αE loop residues (1212−1222).

Figure 2. Distribution of B'-factors for ALK (green), ALKC1156Y (gray) and L1196M (orange) for all heavy atoms with bin size of 0.1 units.

Figure 2 shows the distribution of B' values for ALK, ALKC1156Y, and ALK-L1196M. By definition, the average trends toward 0.0 and values above 0.0 show increased motion while those below 0.0 exhibit decreased motion. While ALK (green) shows a more central tendency toward 0.0, both mutants extend the distribution in both negative and positive directions. The relative B-factors values above 1.75 suggest more dynamic motion for the mutants. Interestingly, the mutants also show regions that exhibit less motion than WT. Although this spreading of the distribution could represent a more active signature, we were eager to examine the more nuanced nature of the B'-factor differences.

Figure 3. Plot of ∆B' values for C1156Y (gray) and L1196M (orange) ALK KD by residue Cα atom. Gaps represent unresolved protein regions.

Using WT ALK as a baseline, ∆B' vales were generated by subtracting the WT B' value from each mutant B' value. ∆B' values > +0.5 are considered to be significant “warming” of the mutant, or increased disorder, relative to WT. ∆B' values < -0.5 are considered to show considerable “cooling”, or less disorder than WT protein. These differential B' values are depicted in Figure 3 for C1156Y (gray) and L1196M (orange). While there are many regions that show ∆B' > +0.5, including the P-loop, αC-helix, αD-αE-loop, β3-αC-loop and the A-loop, there are essentially no regions with ∆B' < -0.5. This data suggests that there are only significant increases in B', or motion, for the mutants relative to WT protein. Meaningfully “warmed” regions occur in the αAL-helix at the base of the Aloop, consisting of residues 1272−1279. Other regions with ∆B' > +0.5 include P-loop residues (1121−1129), αC-helix and

Figure 4. Normalized B-factor differences of ALK mutants relative to wildtype. Residues with ∆B' > +0.5 units (Cα atoms) are colored red for “warming” and ∆B' < -0.5 units (Cα atoms) are colored light blue for “cooling” relative to WT (green); Top panel: C1156Y (gray) apo structure. Bottom panel: L1196M (orange) apo structure.

Figure 4 shows the superposition of WT and mutant structures with regions colored red (∆B' > +0.5) and light blue (∆B' < -0.5). The top panel shows WT and ALK-C1156Y. The C1156Y mutation is located on the β3-αC-loop. The P-loop defines the upper border of the ATP-binding pocket and the tip extends to the A-loop, αC-helix, approaching the C1156Y side chain. At the base, the A-loop sits between the tip of the


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P-loop and the αC-helix and then extends out toward solvent. The αC-helix completes the triad, with residues having close contacts with C1156Y, the tip of the P-loop and the A-loop. Overall, there is very little difference in backbone position of ALK and ALK-C1156Y. The most significant difference in the coordinates is a backbone translation of up to 1.5 Å for residues adjacent to the mutation site (1153−1159). In addition, P-loop residues 1122−1125 are unresolved in the mutant protein. The bottom panel of Figure 4 shows the superposition of WT and L1196M ALK structures. The gatekeeper mutant is located at the back of the ATP binding site in the hinge region (mutant methionine is bolded). Overall, ALK-L1196M shows insignificant backbone positional difference with WT. Additionally; both proteins are similarly resolved with the exception of residue G1123 of the P-loop, which is unresolved in ALK-L1196M. Both activating mutations are distinct in location and properties. While L1196M is a subtle structural residue change from leucine to methionine, C1156Y is more dramatic. Despite the uniqueness of each mutation, both give similar ∆B' fingerprints. The P-loop, αC-helix and A-loop triad contain the most consistent changes of the two mutants. Interestingly, R1275Q, located on the A-loop of ALK, is found in neuroblastoma patients and increased catalytic efficiency of unphosphorylated protein by over 10-fold.13 Structural data revealed a more extended A-loop conformation consistent with a more active conformation.34 Many of the high normalized B-factor differential “hot” spot regions of the mutant proteins overlap with regions of the protein that have higher normalized B-factors in the WT protein. Consequently, more rigid regions in WT ALK tend to stay rigid, while flexible regions have higher tendency to show larger differences in normalized B-factors with activating mutations.

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Figure 5. Normalized B-factor differences of ALK mutants relative to wildtype. Residues with ∆B' > +0.5 units (Cα atoms) are colored red. Top panel: Close up view of stabilizing ‘hydrophobic cluster’ residues for WT (green) and ALK-C1156Y KD (gray). Bottom panel: close up view of WT (green) and ALKL1196M (orange).

A closer view of the C1156Y mutation and surrounding residues is shown in Figure 5 (top panel). A cluster of hydrophobic residues surround F1164, from the αC-helix to the P-loop. The mutant tyrosine in C1156Y displaces F1127 destabilizing the P-loop, rendering it unresolved. Since F1127 is highly conserved among protein kinases (67% F; 23% Y), it may help stabilize the P-loop and hydrophobic core made up of P1153, L1152, F1164 and L1187. Disruption of this stabilized network of lipophilic side chains may be responsible for the increased catalytic activity. This disruption hypothesis is corroborated by the sharp increase in normalized B-factors with the C1156Y mutation. The nearby site L1152R is another common gain-of-function mutation reported in both NSCLC and neuroblastoma. L1152 is part of this hydrophobic cluster and packs against core residue F1164. Apo ALK-L1152R crystallization attempts were unsuccessful and an ALKL1152R-crizotinib complex co-crystal structure (unpublished data) showed an overall decrease in the electron density quality and high disorder of the P-loop residues 1124−1127 and residues 1152−1157. These observations suggest that L1152R also increases protein dynamics, perhaps even to a greater degree than C1156Y. The impact on nearby residues for the L1196M mutant appear more subtle (Figure 5; bottom panel). L1196 is adjacent to I1194, F1164, F1127 and L1187. F1164 forms the center of the aforementioned ‘hydrophobic cluster’ and may be important in the stability of the surrounding secondary motifs. Altogether, these hydrophic residues stem from the αC-helix, β4-β5 sheets and P-loop. In addition, the L1196M mutation is adjacent to I1171, F1271, F1174, and F1245 at the base of the αC-helix. Since I1171 shows a unique conformation relative to WT protein and is adjacent to the DFG sequence at the start of the activation segment, there may be impact on dynamics supported by the increased normalized B-factors beginning at


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ACS Medicinal Chemistry Letters M1273. A previously reported hydrophobic core is centered on F1271 of the conserved DFG residues.35-36 Side chain residues within 4.5 Å of F1271 include I1170/1171/1268/1279, F1174/1245, L1240, H1247 and A1274. Reported mutations in this cluster include F1174, F1245, and I1171T/N/S. These two hydrophobic clusters centered on F1164 and F1271 are part of and connected by the Regulatory-Spine (R-spine, Figure S1).37 Molecular dynamics simulations have been used to predict regions of mobility for ALK mutants, including M1166R, I1171N, F1174L, F1245C, I1250T, G1128A, T1151M, R1192P, and R1275Q in the context of neuroblastoma primary activating mutations. The authors concluded that activating mutations further increase flexibility of the A-loop and Nterminal β-turn and the P-loop and confirm their role in activation. Ultimately these series of changes result in the increased mobility of the A-loop displacing it in a more active position. Ji and co-workers also performed molecular dynamics simulations on the resistant mutation ALK-C1156Y bound to crizotinib.38 They conclude that, although the mutation does not contact crizotinib, changes in nearby protein motifs decrease van der Waals and electrostatic interactions inducing crizotinib displacement. To corroborate normalized B-factor analysis, molecular dynamics (MD) simulations were performed for ALK, ALK-C1156Y, and ALK-L1196M. To simplify and align with the normalized B-factor analysis, only Cα atoms were extracted for investigation in the simulation. Figure S2 highlights the RMSF values by residue. Four key regions of the protein (activation segment, β3-αC-loop, P-loop and αDαE-loop) show the least motion for wildtype protein relative to mutants, consistent with the normalized B-factor analysis. To provide an additional comparison of C1156Y and L1196M structural dynamics relative to wild-type ALK, protein-deuterium exchange experiments were performed. Hydrogen deuterium exchange (HDX) is a reaction that replaces deuterium atoms with hydrogen atoms, or vice versa. Proteins in solution are dynamic and allow exchange reactions to occur on solvent (D2O) exposed regions. Based on the rate of deuterium incorporation, correlations with protein and nucleic acid dynamics have been proposed.39 For both mutants relative to wildtype ALK (Figure S3), HDX results showed enhanced deuterium incorporation in representative P-loop (1119−1137, 1122−1139), αC-helix (1149−1164, 1149−1162) and activation loop (1276−1282, 1277−1282) containing peptides, indicating higher dynamics in these regions for the mutants. Three different peptides were also used as negative controls and showed no differential incorporation of deuterium (Figure S4). In a similar analysis, Hoofnagle and coworkers studied hydrogen/deuterium exchange/mass spectrometry on ERK and pERK showing enhanced incorporation of deuterium in regions of the protein that were both consistent and inconsistent with x-ray coordinate data.40 B-factor data were not discussed in their analysis of the x-ray data. Normalized B-factors are an under-utilized parameter in crystallography, but can be extremely useful since most crystal structures provide atomic level resolution. To compare Bfactors across crystallographic platforms, a normalized Bfactor metric was used. We demonstrated the utility of this method to analyze protein dynamics by selecting simple systems with publically available, high resolution x-ray crystal data. Normalized B-factor evaluation for ALK-WT, ALKC1156Y and ALK-L1196M proteins were used to show flexibility differences and provide mechanistic insight into the activation of quiescent ALK. Differences in mobility were

most pronounced in the P-loops, αC-helices, β3-αC-loops and A-loops. Mutations that disrupt the F1164 or F1271 hydrophobic cores or the stability of the P-loop, αC-helix, β3-αCloop or A-loop may decrease stability. ALK kinetics data and three methods were used to show that selected clinical mutations are activating by increasing protein dynamics relative to wildtype ALK as baseline. Normalized B-factors add another dimension to protein crystal structures that can help distinguish two proteins that look similar by atomic position, provide protein dynamics and inform structural and mechanistic insight into protein function.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental and computational methods. Structural data PDB information.(PDF)

AUTHOR INFORMATION Corresponding Author * email: [email protected].

Author Contributions All authors contributed to this manuscript.

REFERENCES 1. Roskoski, R., Jr. Anaplastic lymphoma kinase (ALK): structure, oncogenic activation, and pharmacological inhibition. Pharmacol Res 2013, 68 (1), 68-94. 2. Soda, M.; Choi, Y. L.; Enomoto, M.; Takada, S.; Yamashita, Y.; Ishikawa, S.; Fujiwara, S.; Watanabe, H.; Kurashina, K.; Hatanaka, H.; Bando, M.; Ohno, S.; Ishikawa, Y.; Aburatani, H.; Niki, T.; Sohara, Y.; Sugiyama, Y.; Mano, H. Identification of the transforming EML4ALK fusion gene in non-small-cell lung cancer. Nature 2007, 448 (7153), 561-6. 3. Holla, V. R.; Elamin, Y. Y.; Bailey, A. M.; Johnson, A. M.; Litzenburger, B. C.; Khotskaya, Y. B.; Sanchez, N. S.; Zeng, J.; Shufean, M. A.; Shaw, K. R.; Mendelsohn, J.; Mills, G. B.; Meric-Bernstam, F.; Simon, G. R. ALK: a tyrosine kinase target for cancer therapy. Cold Spring Harb Mol Case Stud 2017, 3 (1), a001115. 4. Mosse, Y. P.; Laudenslager, M.; Longo, L.; Cole, K. A.; Wood, A.; Attiyeh, E. F.; Laquaglia, M. J.; Sennett, R.; Lynch, J. E.; Perri, P.; Laureys, G.; Speleman, F.; Kim, C.; Hou, C.; Hakonarson, H.; Torkamani, A.; Schork, N. J.; Brodeur, G. M.; Tonini, G. P.; Rappaport, E.; Devoto, M.; Maris, J. M. Identification of ALK as a major familial neuroblastoma predisposition gene. Nature 2008, 455 (7215), 930-5. 5. Schleiermacher, G.; Javanmardi, N.; Bernard, V.; Leroy, Q.; Cappo, J.; Rio Frio, T.; Pierron, G.; Lapouble, E.; Combaret, V.; Speleman, F.; de Wilde, B.; Djos, A.; Ora, I.; Hedborg, F.; Trager, C.; Holmqvist, B. M.; Abrahamsson, J.; Peuchmaur, M.; Michon, J.; Janoueix-Lerosey, I.; Kogner, P.; Delattre, O.; Martinsson, T. Emergence of new ALK mutations at relapse of neuroblastoma. J Clin Oncol 2014, 32 (25), 2727-34. 6. Murugan, A. K.; Xing, M. Anaplastic thyroid cancers harbor novel oncogenic mutations of the ALK gene. Cancer Res 2011, 71 (13), 4403-11. 7. Cui, J. J.; Tran-Dube, M.; Shen, H.; Nambu, M.; Kung, P. P.; Pairish, M.; Jia, L.; Meng, J.; Funk, L.; Botrous, I.; McTigue, M.; Grodsky, N.; Ryan, K.; Padrique, E.; Alton, G.; Timofeevski, S.; Yamazaki, S.; Li, Q.; Zou, H.; Christensen, J.; Mroczkowski, B.; Bender, S.; Kania, R. S.; Edwards, M. P. Structure based drug design of crizotinib (PF-02341066), a potent and selective dual inhibitor of mesenchymalepithelial transition factor (c-MET) kinase and anaplastic lymphoma kinase (ALK). J Med Chem 2011, 54 (18), 6342-63. 8. Choi, Y. L.; Soda, M.; Yamashita, Y.; Ueno, T.; Takashima, J.; Nakajima, T.; Yatabe, Y.; Takeuchi, K.; Hamada, T.; Haruta, H.; Ishikawa, Y.; Kimura, H.; Mitsudomi, T.; Tanio, Y.; Mano, H.; Group, A.


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L. K. L. C. S. EML4-ALK mutations in lung cancer that confer resistance to ALK inhibitors. N Engl J Med 2010, 363 (18), 1734-9. 9. Katayama, R.; Friboulet, L.; Koike, S.; Lockerman, E. L.; Khan, T. M.; Gainor, J. F.; Iafrate, A. J.; Takeuchi, K.; Taiji, M.; Okuno, Y.; Fujita, N.; Engelman, J. A.; Shaw, A. T. Two novel ALK mutations mediate acquired resistance to the next-generation ALK inhibitor alectinib. Clin Cancer Res 2014, 20 (22), 5686-96. 10. Katayama, R.; Khan, T. M.; Benes, C.; Lifshits, E.; Ebi, H.; Rivera, V. M.; Shakespeare, W. C.; Iafrate, A. J.; Engelman, J. A.; Shaw, A. T. Therapeutic strategies to overcome crizotinib resistance in non-small cell lung cancers harboring the fusion oncogene EML4-ALK. Proc Natl Acad Sci U S A 2011, 108 (18), 7535-40. 11. Katayama, R.; Shaw, A. T.; Khan, T. M.; Mino-Kenudson, M.; Solomon, B. J.; Halmos, B.; Jessop, N. A.; Wain, J. C.; Yeo, A. T.; Benes, C.; Drew, L.; Saeh, J. C.; Crosby, K.; Sequist, L. V.; Iafrate, A. J.; Engelman, J. A. Mechanisms of acquired crizotinib resistance in ALKrearranged lung Cancers. Sci Transl Med 2012, 4 (120), 120ra17. 12. Gainor, J. F.; Dardaei, L.; Yoda, S.; Friboulet, L.; Leshchiner, I.; Katayama, R.; Dagogo-Jack, I.; Gadgeel, S.; Schultz, K.; Singh, M.; Chin, E.; Parks, M.; Lee, D.; DiCecca, R. H.; Lockerman, E.; Huynh, T.; Logan, J.; Ritterhouse, L. L.; Le, L. P.; Muniappan, A.; Digumarthy, S.; Channick, C.; Keyes, C.; Getz, G.; Dias-Santagata, D.; Heist, R. S.; Lennerz, J.; Sequist, L. V.; Benes, C. H.; Iafrate, A. J.; Mino-Kenudson, M.; Engelman, J. A.; Shaw, A. T. Molecular Mechanisms of Resistance to First- and Second-Generation ALK Inhibitors in ALK-Rearranged Lung Cancer. Cancer Discov 2016, 6 (10), 1118-1133. 13. Bresler, S. C.; Weiser, D. A.; Huwe, P. J.; Park, J. H.; Krytska, K.; Ryles, H.; Laudenslager, M.; Rappaport, E. F.; Wood, A. C.; McGrady, P. W.; Hogarty, M. D.; London, W. B.; Radhakrishnan, R.; Lemmon, M. A.; Mosse, Y. P. ALK mutations confer differential oncogenic activation and sensitivity to ALK inhibition therapy in neuroblastoma. Cancer Cell 2014, 26 (5), 682-94. 14. Huang, W. S.; Liu, S.; Zou, D.; Thomas, M.; Wang, Y.; Zhou, T.; Romero, J.; Kohlmann, A.; Li, F.; Qi, J.; Cai, L.; Dwight, T. A.; Xu, Y.; Xu, R.; Dodd, R.; Toms, A.; Parillon, L.; Lu, X.; Anjum, R.; Zhang, S.; Wang, F.; Keats, J.; Wardwell, S. D.; Ning, Y.; Xu, Q.; Moran, L. E.; Mohemmad, Q. K.; Jang, H. G.; Clackson, T.; Narasimhan, N. I.; Rivera, V. M.; Zhu, X.; Dalgarno, D.; Shakespeare, W. C. Discovery of Brigatinib (AP26113), a Phosphine Oxide-Containing, Potent, Orally Active Inhibitor of Anaplastic Lymphoma Kinase. J Med Chem 2016, 59 (10), 4948-64. 15. Kinoshita, K.; Kobayashi, T.; Asoh, K.; Furuichi, N.; Ito, T.; Kawada, H.; Hara, S.; Ohwada, J.; Hattori, K.; Miyagi, T.; Hong, W. S.; Park, M. J.; Takanashi, K.; Tsukaguchi, T.; Sakamoto, H.; Tsukuda, T.; Oikawa, N. 9-substituted 6,6-dimethyl-11-oxo-6,11-dihydro-5Hbenzo[b]carbazoles as highly selective and potent anaplastic lymphoma kinase inhibitors. J Med Chem 2011, 54 (18), 6286-94. 16. Marsilje, T. H.; Pei, W.; Chen, B.; Lu, W.; Uno, T.; Jin, Y.; Jiang, T.; Kim, S.; Li, N.; Warmuth, M.; Sarkisova, Y.; Sun, F.; Steffy, A.; Pferdekamper, A. C.; Li, A. G.; Joseph, S. B.; Kim, Y.; Liu, B.; Tuntland, T.; Cui, X.; Gray, N. S.; Steensma, R.; Wan, Y.; Jiang, J.; Chopiuk, G.; Li, J.; Gordon, W. P.; Richmond, W.; Johnson, K.; Chang, J.; Groessl, T.; He, Y. Q.; Phimister, A.; Aycinena, A.; Lee, C. C.; Bursulaya, B.; Karanewsky, D. S.; Seidel, H. M.; Harris, J. L.; Michellys, P. Y. Synthesis, structure-activity relationships, and in vivo efficacy of the novel potent and selective anaplastic lymphoma kinase (ALK) inhibitor 5chloro-N2-(2-isopropoxy-5-methyl-4-(piperidin-4-yl)phenyl)-N4-(2(isopropylsulf onyl)phenyl)pyrimidine-2,4-diamine (LDK378) currently in phase 1 and phase 2 clinical trials. J Med Chem 2013, 56 (14), 5675-90. 17. Johnson, T. W.; Richardson, P. F.; Bailey, S.; Brooun, A.; Burke, B. J.; Collins, M. R.; Cui, J. J.; Deal, J. G.; Deng, Y. L.; Dinh, D.; Engstrom, L. D.; He, M.; Hoffman, J.; Hoffman, R. L.; Huang, Q.; Kania, R. S.; Kath, J. C.; Lam, H.; Lam, J. L.; Le, P. T.; Lingardo, L.; Liu, W.; McTigue, M.; Palmer, C. L.; Sach, N. W.; Smeal, T.; Smith, G. L.; Stewart, A. E.; Timofeevski, S.; Zhu, H.; Zhu, J.; Zou, H. Y.; Edwards, M. P. Discovery of (10R)-7-amino-12-fluoro-2,10,16-trimethyl-15-oxo10,15,16,17-tetrahydro-2H-8,4-(m etheno)pyrazolo[4,3-h][2,5,11]benzoxadiazacyclotetradecine-3-carbonitrile (PF-06463922), a macrocyclic inhibitor of anaplastic lymphoma kinase (ALK) and c-ros oncogene 1 (ROS1) with preclinical brain exposure and broad-spectrum potency against ALK-resistant mutations. J Med Chem 2014, 57 (11), 4720-44. 18. Tartari, C. J.; Gunby, R. H.; Coluccia, A. M.; Sottocornola, R.; Cimbro, B.; Scapozza, L.; Donella-Deana, A.; Pinna, L. A.; GambacortiPasserini, C. Characterization of some molecular mechanisms governing autoactivation of the catalytic domain of the anaplastic lymphoma kinase. J Biol Chem 2008, 283 (7), 3743-50.

Page 6 of 7

19. Ringe, D.; Petsko, G. A. Study of protein dynamics by X-ray diffraction. Methods Enzymol 1986, 131, 389-433. 20. Huse, M.; Kuriyan, J. The conformational plasticity of protein kinases. Cell 2002, 109 (3), 275-82. 21. Choy, M. S.; Li, Y.; Machado, L.; Kunze, M. B. A.; Connors, C. R.; Wei, X.; Lindorff-Larsen, K.; Page, R.; Peti, W. Conformational Rigidity and Protein Dynamics at Distinct Timescales Regulate PTP1B Activity and Allostery. Mol Cell 2017, 65 (4), 644-658 e5. 22. Carroll, M. J.; Mauldin, R. V.; Gromova, A. V.; Singleton, S. F.; Collins, E. J.; Lee, A. L. Evidence for dynamics in proteins as a mechanism for ligand dissociation. Nat Chem Biol 2012, 8 (3), 246-52. 23. Henzler-Wildman, K. A.; Lei, M.; Thai, V.; Kerns, S. J.; Karplus, M.; Kern, D. A hierarchy of timescales in protein dynamics is linked to enzyme catalysis. Nature 2007, 450 (7171), 913-6. 24. Nagel, Z. D.; Dong, M.; Bahnson, B. J.; Klinman, J. P. Impaired protein conformational landscapes as revealed in anomalous Arrhenius prefactors. Proc Natl Acad Sci U S A 2011, 108 (26), 10520-5. 25. Tzeng, S. R.; Kalodimos, C. G. Protein activity regulation by conformational entropy. Nature 2012, 488 (7410), 236-40. 26. Campbell, E.; Kaltenbach, M.; Correy, G. J.; Carr, P. D.; Porebski, B. T.; Livingstone, E. K.; Afriat-Jurnou, L.; Buckle, A. M.; Weik, M.; Hollfelder, F.; Tokuriki, N.; Jackson, C. J. The role of protein dynamics in the evolution of new enzyme function. Nat Chem Biol 2016, 12 (11), 944-950. 27. Johnson, L. N.; Noble, M. E.; Owen, D. J. Active and inactive protein kinases: structural basis for regulation. Cell 1996, 85 (2), 149-58. 28. Adams, J. A. Activation loop phosphorylation and catalysis in protein kinases: is there functional evidence for the autoinhibitor model? Biochemistry 2003, 42 (3), 601-7. 29. Schindler, T.; Bornmann, W.; Pellicena, P.; Miller, W. T.; Clarkson, B.; Kuriyan, J. Structural mechanism for STI-571 inhibition of abelson tyrosine kinase. Science 2000, 289 (5486), 1938-42. 30. Nolen, B.; Taylor, S.; Ghosh, G. Regulation of protein kinases; controlling activity through activation segment conformation. Mol Cell 2004, 15 (5), 661-75. 31. Wan, P. T.; Garnett, M. J.; Roe, S. M.; Lee, S.; NiculescuDuvaz, D.; Good, V. M.; Jones, C. M.; Marshall, C. J.; Springer, C. J.; Barford, D.; Marais, R. Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell 2004, 116 (6), 855-67. 32. Lu, W. C.; Wang, C. Z.; Yu, E. W.; Ho, K. M. Dynamics of the trimeric AcrB transporter protein inferred from a B-factor analysis of the crystal structure. Proteins 2006, 62 (1), 152-8. 33. Siglioccolo, A.; Gerace, R.; Pascarella, S. "Cold spots" in protein cold adaptation: Insights from normalized atomic displacement parameters (B'-factors). Biophys Chem 2010, 153 (1), 104-14. 34. Epstein, L. F.; Chen, H.; Emkey, R.; Whittington, D. A. The R1275Q neuroblastoma mutant and certain ATP-competitive inhibitors stabilize alternative activation loop conformations of anaplastic lymphoma kinase. J Biol Chem 2012, 287 (44), 37447-57. 35. Bossi, R. T.; Saccardo, M. B.; Ardini, E.; Menichincheri, M.; Rusconi, L.; Magnaghi, P.; Orsini, P.; Avanzi, N.; Borgia, A. L.; Nesi, M.; Bandiera, T.; Fogliatto, G.; Bertrand, J. A. Crystal structures of anaplastic lymphoma kinase in complex with ATP competitive inhibitors. Biochemistry 2010, 49 (32), 6813-25. 36. Lee, C. C.; Jia, Y.; Li, N.; Sun, X.; Ng, K.; Ambing, E.; Gao, M. Y.; Hua, S.; Chen, C.; Kim, S.; Michellys, P. Y.; Lesley, S. A.; Harris, J. L.; Spraggon, G. Crystal structure of the ALK (anaplastic lymphoma kinase) catalytic domain. Biochem J 2010, 430 (3), 425-37. 37. Shaw, A. S.; Kornev, A. P.; Hu, J.; Ahuja, L. G.; Taylor, S. S. Kinases and pseudokinases: lessons from RAF. Mol Cell Biol 2014, 34 (9), 1538-46. 38. Sun, H. Y.; Ji, F. Q. A molecular dynamics investigation on the crizotinib resistance mechanism of C1156Y mutation in ALK. Biochem Biophys Res Commun 2012, 423 (2), 319-24. 39. Englander, S. W.; Kallenbach, N. R. Hydrogen exchange and structural dynamics of proteins and nucleic acids. Q Rev Biophys 1983, 16 (4), 521-655. 40. Hoofnagle, A. N.; Resing, K. A.; Goldsmith, E. J.; Ahn, N. G. Changes in protein conformational mobility upon activation of extracellular regulated protein kinase-2 as detected by hydrogen exchange. Proc Natl Acad Sci U S A 2001, 98 (3), 956-61.


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Reviving B-Factors: Activating ALK Mutations Increase Protein

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