Osimertinib has been demonstrated to overcome the epidermal growth factor receptor (EGFR)-T790M, the most relevant acquired resistance to first-generation EGFR–tyrosine kinase inhibitors (EGFR–TKIs). However, the C797S mutation, which impairs the covalent binding between the cysteine residue at position 797 of EGFR and osimertinib, induces resistance to osimertinib. Currently, there are no effective therapeutic strategies to overcome the C797S/T790M/activating-mutation (triple-mutation)-mediated EGFR–TKI resistance. In the present study, we identify brigatinib to be effective against triple-mutation-harbouring cells in vitro and in vivo. Our original computational simulation demonstrates that brigatinib fits into the ATP-binding pocket of triple-mutant EGFR. The structure–activity relationship analysis reveals the key component in brigatinib to inhibit the triple-mutant EGFR. The efficacy of brigatinib is enhanced markedly by combination with anti-EGFR antibody because of the decrease of surface and total EGFR expression. Thus, the combination therapy of brigatinib with anti-EGFR antibody is a powerful candidate to overcome triple-mutant EGFR.

Mutational activation of the Ras oncogene products (H-Ras, K-Ras, and N-Ras) is frequently observed in human cancers, making them promising anticancer drug targets. Nonetheless, no effective strategy has been available for the development of Ras inhibitors, partly owing to the absence of well-defined surface pockets suitable for drug binding. Only recently, such pockets have been found in the crystal structures of a unique conformation of Ras⋅GTP. Here we report the successful development of small-molecule Ras inhibitors by an in silico screen targeting a pocket found in the crystal structure of M-Ras⋅GTP carrying an H-Ras–type substitution P40D. The selected compound Kobe0065 and its analog Kobe2602 exhibit inhibitory activity toward H-Ras⋅GTP-c-Raf-1 binding both in vivo and in vitro. They effectively inhibit both anchorage-dependent and -independent growth and induce apoptosis of H- ras G12V –transformed NIH 3T3 cells, which is accompanied by down-regulation of downstream molecules such as MEK/ERK, Akt, and RalA as well as an upstream molecule, Son of sevenless. Moreover, they exhibit antitumor activity on a xenograft of human colon carcinoma SW480 cells carrying the K-ras G12V gene by oral administration. The NMR structure of a complex of the compound with H-Ras⋅GTP T35S , exclusively adopting the unique conformation, confirms its insertion into one of the surface pockets and provides a molecular basis for binding inhibition toward multiple Ras⋅GTP-interacting molecules. This study proves the effectiveness of our strategy for structure-based drug design to target Ras⋅GTP, and the resulting Kobe0065-family compounds may serve as a scaffold for the development of Ras inhibitors with higher potency and specificity.

Ras family small GTPases assume two interconverting conformations, "inactive" state 1 and "active" state 2, in their GTPbound forms. Here, to clarify the mechanism of state transition, we have carried out x-ray crystal structure analyses of a series of mutant H-Ras and M-Ras in complex with guanosine 5-(␤,␥-imido)triphosphate (GppNHp), representing various intermediate states of the transition. Crystallization of H-RasT35S-GppNHp enables us to solve the first complete tertiary structure of H-Ras state 1 possessing two surface pockets unseen in the state 2 or H-Ras-GDP structure. Moreover, determination of the two distinct crystal structures of H-RasT35S-GppNHp, showing prominent polysterism in the switch I and switch II regions, reveals a pivotal role of the guanine nucleotide-mediated interaction between the two switch regions and its rearrangement by a nucleotide positional change in the state 2 to state 1 transition. Furthermore, the 31 P NMR spectra and crystal structures of the GppNHp-bound forms of M-Ras mutants, carrying various H-Ras-type amino acid substitutions, also reveal the existence of a surface pocket in state 1 and support a similar mechanism based on the nucleotide-mediated interaction and its rearrangement in the state 1 to state 2 transition. Intriguingly, the conformational changes accompanying the state transition mimic those that occurred upon GDP/GTP exchange, indicating a common mechanistic basis inherent in the high flexibility of the switch regions. Collectively, these results clarify the structural features distinguishing the two states and provide new insights into the molecular basis for the state transition of Ras protein.Small GTPases Ras (H-Ras, K-Ras, and N-Ras) are the products of the ras proto-oncogenes and presumed to be some of the most promising targets for anti-cancer drug development because of their high frequency of mutational activation in a variety of human cancers (1). Ras functions as a molecular switch by cycling between GTP-bound active and GDP-bound inactive forms in intracellular signaling pathways controlling cell growth and differentiation. Conversion between the GDPbound and the GTP-bound forms is controlled by guanine nucleotide exchange factors and GTPase-activating proteins (2, 3). Ras comprises the Ras family of small GTPases together with a number of its relatives, including Rap1, Rap2, R-Ras, R-Ras2/ TC1, M-Ras/R-Ras3, etc. (1). X-ray crystallographic and NMR analyses of H-Ras and Rap1A, alone or in complex with their effectors, revealed that the exchange of GTP for GDP results in allosteric conformational changes in two adjacent regions, termed switch I (residues 32-38) and switch II (residues 60 -75), and enables Ras to execute downstream signaling through direct interaction with its effectors, such as Raf kinases and phosphoinositide 3-kinases (2, 3). Recent 31 P NMR spectroscopic studies on Ras unveiled its novel structural feature, the conformational dynamics in the GTP-bound form (4). H-Ras and K-Ras in complex with Mg 2ϩ and a non-hydrolyzable GT...

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Ras small GTPases undergo dynamic equilibrium of two interconverting conformations, state 1 and state 2, in the GTPbound forms, where state 2 is recognized by effectors, whereas physiological functions of state 1 have been unknown. Limited information, such as static crystal structures and 31 P NMR spectra, was available for the study of the conformational dynamics. Here we determine the solution structure and dynamics of state 1 by multidimensional heteronuclear NMR analysis of an HRasT35S mutant in complex with guanosine 5-(␤, ␥-imido)-triphosphate (GppNHp). The state 1 structure shows that the switch I loop fluctuates extensively compared with that in state 2 or H-Ras-GDP. Also, backbone 1 H, 15 N signals for state 2 are identified, and their dynamics are studied by utilizing a complex with c-Raf-1. Furthermore, the signals for almost all the residues of H-Ras⅐GppNHp are identified by measurement at low temperature, and the signals for multiple residues are found split into two peaks corresponding to the signals for state 1 and state 2. Intriguingly, these residues are located not only in the switch regions and their neighbors but also in the rigidly structured regions, suggesting that global structural rearrangements occur during the state interconversion. The backbone dynamics of each state show that the switch loops in state 1 are dynamically mobile on the picosecond to nanosecond time scale, and these mobilities are significantly reduced in state 2. These results suggest that multiconformations existing in state 1 are mostly deselected upon the transition toward state 2 induced by the effector binding.Small GTPases H-Ras, K-Ras, and N-Ras, collectively called Ras, are the products of the ras proto-oncogenes and function as molecular switches by cycling between the GTP-bound active and the GDP-bound inactive forms in intracellular signaling pathways controlling proliferation, differentiation, and apoptosis of cells. GTP hydrolysis on Ras is markedly stimulated by GTPase-activating proteins, whereas conversion from the GDP-bound form to the GTP-bound form is promoted by guanine nucleotide exchange factors (1, 2). Ras comprise the Ras family of small GTPases together with a number of its relatives including Rap1, Rap2, R-Ras, R-Ras2/TCL, M-Ras/RRas3, RalA, RalB, etc. (3). Structural studies of Ras showed that structural differences between the GDP-and GTP-bound forms universally exist in two flexible regions, called switch I (residues 32-38 in H-Ras) and switch II (residues 60 -75 in H-Ras) (1). GTP-sensitive orientation of the switch regions enables Ras to interact with their effectors such as Raf kinases and phosphoinositide 3-kinases (2). Recent 31 P NMR studies suggested that H-Ras in the nucleoside triphosphate form exists in equilibrium between two kinds of conformational states, state 1 and state 2, around the phosphate groups of GTP or its non-hydrolyzable analogues, GppNHp 3 and GTP␥S, bound to the protein (4 -6). This conformational heterogeneity has been commonly observed in a number of Ras homolo...

Background Alectinib has shown a greater efficacy to ALK -rearranged non-small-cell lung cancers in first-line setting; however, most patients relapse due to acquired resistance, such as secondary mutations in ALK including I1171N and G1202R. Although ceritinib or lorlatinib was shown to be effective to these resistant mutants, further resistance often emerges due to ALK-compound mutations in relapse patients following the use of ceritinib or lorlatinib. However, the drug for overcoming resistance has not been established yet. Methods We established lorlatinib-resistant cells harboring ALK-I1171N or -G1202R compound mutations by performing ENU mutagenesis screening or using an in vivo mouse model. We performed drug screening to overcome the lorlatinib-resistant ALK-compound mutations. To evaluate these resistances in silico , we developed a modified computational molecular dynamic simulation (MP-CAFEE). Findings We identified 14 lorlatinib-resistant ALK-compound mutants, including several mutants that were recently discovered in lorlatinib-resistant patients. Some of these compound mutants were found to be sensitive to early generation ALK-TKIs and several BCR-ABL inhibitors. Using our original computational simulation, we succeeded in demonstrating a clear linear correlation between binding free energy and in vitro experimental IC 50 value of several ALK-TKIs to single- or compound-mutated EML4-ALK expressing Ba/F3 cells and in recapitulating the tendency of the binding affinity reduction by double mutations found in this study. Computational simulation revealed that ALK-L1256F single mutant conferred resistance to lorlatinib but increased the sensitivity to alectinib. Interpretation We discovered lorlatinib-resistant multiple ALK-compound mutations and an L1256F single mutation as well as the potential therapeutic strategies for these ALK mutations. Our original computational simulation to calculate the binding affinity may be applicable for predicting resistant mutations and for overcoming drug resistance in silico. Fund This work was mainly supported by MEXT/JSPS KAKENHI Grants and AMED Grants.

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In order to predict the accurate binding configuration as well as the binding affinity for a flexible protein receptor and its inhibitor drug, enhanced sampling with multicanonical molecular dynamics (McMD) simulation and thermodynamic integration (TI) were combined as a general drug docking method. CDK2, cyclin-dependent kinase 2, is involved in the cell cycle regulation. Malfunctions in CDK2 can cause tumorigenesis, and thus it is a potential drug target. Here, we performed a long McMD simulation for docking the inhibitor CS3 to CDK2 starting from the unbound structure. Subsequently, a potential binding/unbinding pathway was given from the multicanonical ensemble, and the binding free energy was readily computed by TI along the pathway. Using this combination, the correct binding configuration of CS3 to CDK2 was obtained, and its affinity coincided well with the experimental value.