Staurosporine scaffold–based rational discovery of the wild‐type sparing reversible inhibitors of EGFR T790M gatekeeper mutant in lung cancer with analog‐sensitive kinase technology

The human epidermal growth factor receptor (EGFR) has been established as an attractive target for lung cancer therapy. However, an acquired EGFR T790M gatekeeper mutation is frequently observed in patients treated with first‐line anticancer agents such as gefitinib and erlotinib to cause drug resistance, largely limiting the application of small‐molecule kinase inhibitors in EGFR‐targeted chemotherapy. Previously, the reversible pan‐kinase inhibitor staurosporine and its several analogs such as Gö6976 and K252a have been reported to selectively inhibit the EGFR T790M mutant (EGFR^T790M) over wild‐type kinase (EGFR^WT), suggesting that the staurosporine scaffold is potentially to develop the wild‐type sparing reversible inhibitors of EGFR^T790M. Here, we systematically evaluated the inhibitor response of 28 staurosporine scaffold–based compounds to EGFR T790M mutation at structural, energetic, and molecular levels by using an integrated in silico–in vitro analog‐sensitive (AS) kinase technology. With the strategy, we were able to identify 4 novel wild‐type sparing inhibitors UCN‐01, UCN‐02, AFN941, and SB‐218078 with high or moderate selectivity of 30‐, 45‐, 5‐, and 8‐fold for EGFR^T790M over EGFR^WT, respectively, which are comparable with or even better than that of the parent compound staurosporine (24‐fold). Molecular modeling and structural analysis revealed that van der Waals contacts and hydrophobic forces can form between the side chain of mutated residue Met790 and the pyrrolidinone moiety of inhibitor ligand UCN‐02, which may simultaneously improve the favorable interaction energy between the kinase and inhibitor, and reduce the unfavorable desolvation penalty upon the kinase‐inhibitor binding. A hydroxyl group of UCN‐02 additional to staurosporine locates at the pyrrolidinone moiety, which can largely alter the electronic distribution of pyrrolidinone moiety and thus promote the intermolecular interaction with Met790 residue. This can well explain the measured higher selectivity of UCN‐02 than staurosporine for mutant over wild‐type kinase.     

A systematic analysis of the resistance and sensitivity of HER2YVMA receptor tyrosine kinase mutant to tyrosine kinase inhibitors in HER2-positive lung cancer

Human epidermal growth factor receptor 2 (HER2) has become a well-established target for the treatment of HER2-positive lung cancer. However, a frequently observed in-frame mutation that inserts amino acid quadruplex Tyr776-Val777-Met778-Ala779 at G776 (G776^YVMA) in HER2 kinase domain can cause drug resistance and sensitivity, largely limiting the application of reversible tyrosine kinase inhibitors in lung cancer therapy. A systematic investigation of the intermolecular interactions between the HER2^YVMA mutant and clinical small-molecule inhibitors would help to establish a complete picture of drug response to HER2 G776^YVMA insertion in lung cancer, and to design new tyrosine kinase inhibitors with high potency and selectivity to target the lung cancer-related HER2^YVMA mutant. Here, we combined homology modeling, ligand grafting, structure minimization, molecular simulation and binding affinity analysis to profile a number of tyrosine kinase inhibitors against the G776^YVMA insertion in HER2. It is found that the insertion is far away from HER2 active pocket and thus cannot contact inhibitor ligand directly. However, the insertion is expected to induce marked allosteric effect on some regions around the pocket, including A-loop and hinges connecting between the N- and C-lobes of HER2 kinase domain, which may exert indirect influence to inhibitor binding. Most investigated inhibitors exhibit weak binding strength to both wild-type and mutant HER2, which can be attributed to steric hindrance that impairs ligand compatibility with HER2 active pocket. However, the cognate inhibitor lapatinib and the non-cognate inhibitor bosutinib were predicted to have low affinity for wild-type HER2 but high affinity for HER2^YVMA mutant, which was confirmed by subsequent kinase assay experiments; the inhibitory potencies of bosutinib against wild-type and mutant HER2 were determined to be IC₅₀?>?1000 and =27?nM, respectively, suggesting that the bosutinib might be exploited as a selective inhibitor for mutant over wild-type HER2. Structural examination revealed that formation of additional non-bonded interactions such as hydrogen bonds and hydrophobic contacts with HER2 A-loop region due to G776^YVMA insertion is the primary factor to improve bosutinib affinity upon the mutation.     

Click chemistry for improvement in selectivity of quinazoline-based kinase inhibitors for mutant epidermal growth factor receptors

Discovery of mutant-selective kinase inhibitors is one of the challenges in medicinal chemistry and is a main issue for epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors. We tried to improve the selectivity of pan-HER inhibitors for mutant EGFRs. Utilizing click chemistry, triazole-tethered quinazoline derivatives were synthesized, based on a quinazoline scaffold showing pan-HER inhibition. The representative compound 5j exhibited 17- and 52-fold improved selectivity for EGFR L858R/T790M over wild-type EGFR and HER2, respectively, and demonstrated 6.7-fold more potent antiproliferative activity against PC9 cells harboring EGFR-activating mutation than gefitinib. Although the described quinazolines did not surpass pyrimidines as 3rd generation EGFR inhibitors in terms of selectivity for mutant EGFRs, our approach might provide information that would help in the identification of mutant-selective compounds among pan-HER inhibitors using the quinazoline scaffold.     

JAK3 inhibitor VI is a mutant specific inhibitor for epidermal growth factor receptor with the gatekeeper mutation T790M

AIM: To identify non-quinazoline kinase inhibitors effective against drug resistant mutants of epidermal growth factor receptor (EGFR).METHODS: A kinase inhibitor library was subjected to screening for specific inhibition pertaining to the in vitro kinase activation of EGFR with the gatekeeper mutation T790M, which is resistant to small molecular weight tyrosine kinase inhibitors (TKIs) for EGFR in non-small cell lung cancers (NSCLCs). This inhibitory effect was confirmed by measuring autophosphorylation of EGFR T790M/L858R in NCI-H1975 cells, an NSCLC cell line harboring the gatekeeper mutation. The effects of a candidate compound, Janus kinase 3 (JAK3) inhibitor VI, on cell proliferation were evaluated using the MTT assay and were compared between T790M-positive and -negative lung cancer cell lines. JAK3 inhibitor VI was modeled into the ATP-binding pocket of EGFR T790M/L858R. Potential physical interactions between the compound and kinase domains of wild-type (WT) or mutant EGFRs or JAK3 were estimated by calculating binding energy. The gatekeeper residues of EGFRs and JAKs were aligned to discuss the similarities among EGFR T790M and JAKs.RESULTS: We found that JAK3 inhibitor VI, a known inhibitor for JAK3 tyrosine kinase, selectively inhibits EGFR T790M/L858R, but has weaker inhibitory effects on the WT EGFR in vitro. JAK3 inhibitor VI also specifically reduced autophosphorylation of EGFR T790M/L858R in NCI-H1975 cells upon EGF stimulation, but did not show the inhibitory effect on WT EGFR in A431 cells. Furthermore, JAK3 inhibitor VI suppressed the proliferation of NCI-H1975 cells, but showed limited inhibitory effects on the WT EGFR-expressing cell lines A431 and A549. A docking simulation between JAK3 inhibitor VI and the ATP-binding pocket of EGFR T790M/L858R predicted a potential binding status with hydrogen bonds. Estimated binding energy of JAK3 inhibitor VI to EGFR T790M/L858R was more stable than its binding energy to the WT EGFR. Amino acid sequence alignments revealed that the gatekeeper residues of JAK family kinases are methionine in WT, similar to EGFR T790M, suggesting that TKIs for JAKs may also be effective for EGFR T790M.CONCLUSION: Our findings demonstrate that JAK3 inhibitor VI is a gatekeeper mutant selective TKI and offer a strategy to search for new EGFR T790M inhibitors.     

Recent Development of the Second and Third Generation Irreversible Epidermal Growth Factor Receptor Inhibitors

Recent reports suggested that essential directions for new lung cancer, breast carcinoma therapies, as well as the roomier realm of targeted cancer therapies were provided through targeting the epidermal growth factor receptor (EGFR). Patients who carrying non‐small cell lung carcinoma (NSCLC) with activating mutations in EGFR initially respond well to the EGFR inhibitors erlotinib and gefitinib, which were located the active site of the EGFR kinase and designed to act as competitive inhibitors of combining with the ATP. However, patients who were treated with the erlotinib and gefitinib will relapse because of the emergence of drug‐resistant mutations, with T790M mutations accounting for approximately 60% of all resistance. In order to overcome drug resistance, Pharmaceutical chemistry experts recently devoted great endeavors to the development of second‐generation irreversible selective inhibitors which covalently modify Cys797 or Cys773 at the ATP binding cleft. Nevertheless, these inhibitors have not reached ideal effect of experts in patients with T790M positive mutation and apparently because of the dose‐limiting toxicities associated with inhibition of wild type EGFR. A novel class of ‘third generation’ EGFR TKIs have been developed that is sensitising and T790M mutant‐specific whilst sparing WT EGFR, representing a significant breakthrough in the treatment in NSCLC patients with acquired resistance harboring these genotypes. Herein, we provides an overview of the second and third generation inhibitors currently approved, in clinical trial and also encompasses novel structures of discovery. This review mainly focuses on drug resistance, their mechanisms of action, development of structure–activity relationships and binding modes.     

Structure-based design of flavone-based inhibitors of wild-type and T315I mutant of ABL

The existence of drug resistance caused by mutations in the break-point cluster region-Abelson (BCR-ABL) tyrosine kinase domain remains a clinical challenge due to limited treatment options for effective CML therapies. Here, we report a series of flavone-based common inhibitors equipotent for the wild type and the most drug-resistant T315I mutant of BCR-ABL. The original hit 1 was extensively modified through a structure-based drug design strategy, especially by varying the C7 acetamide appendage of the scaffold to exploit extended interactions with P-loop residues. Structural features relevant to the stabilization of the newly identified inhibitors in the ATP-binding site of ABL are discussed in detail.     

Selective RAF inhibitor impairs ERK1/2 phosphorylation and growth in mutant NRAS,vemurafenib‐resistant melanoma cells

The RAF inhibitor vemurafenib achieves remarkable clinical responses in mutant BRAF melanoma patients. However, vemurafenib is burdened by acquired drug resistance and by the side effects associated with its paradoxical activation of the ERK1/2 pathway in wild‐type BRAF cells. This paradoxical effect has driven the development of a new class of RAF inhibitors. Here, we tested one of these selective, non‐paradox‐inducing RAF inhibitors termed paradox‐breaker‐04 (PB04) or PLX7904. Consistent with its design, PB04 is able to efficiently inhibit activation of ERK1/2 in mutant BRAF melanoma cells but does not hyperactivate ERK1/2 in mutant RAS‐expressing cells. Importantly, PB04 inhibited ERK1/2 phosphorylation in mutant BRAF melanoma cells with acquired resistance to vemurafenib/PLX4720 that is mediated by a secondary mutation in NRAS. Consistent with ERK1/2 reactivation driving the re‐acquisition of malignant properties, PB04 promoted apoptosis and inhibited entry into S phase and anchorage‐independent growth in mutant N‐RAS‐mediated vemurafenib‐resistant cells. These data indicate that paradox‐breaker RAF inhibitors may be clinically effective as a second‐line option in a cohort of acquired vemurafenib‐resistant patients.     

Consequences of lysine 72 mutation on the phosphorylation and activation state of cAMP-dependent kinase

General strategies to obtain inactive kinases have utilized mutation of key conserved residues in the kinase core, and the equivalent Lys72 in cAMP-dependent kinase has often been used to generate a "dead" kinase. Here, we have analyzed the consequences of this mutation on kinase structure and function. Mutation of Lys72 to histidine (K72H) generated an inactive enzyme, which was unphosphorylated. Treatment with an exogenous kinase (PDK-1) resulted in a mutant that was phosphorylated only at Thr197 and remained inactive but nevertheless capable of binding ATP. Ser338 in K72H cannot be autophosphorylated, nor can it be phosphorylated in an intermolecular process by active wild type C-subunit. The Lys72 mutant, once phosphorylated on Thr197, can bind with high affinity to the RIalpha subunits. Thus a dead kinase can still act as a scaffold for binding substrates and inhibitors; it is only phosphoryl transfer that is defective. Using a potent inhibitor of C-subunit activity, H-89, Escherichia coli-expressed C-subunit was also obtained in its unphosphorylated state. This protein is able to mature into its active form in the presence of PDK-1 and is able to undergo secondary autophosphorylation on Ser338. Unlike the H-89-treated wild type protein, the mutant protein (K72H) cannot undergo the subsequent cis autophosphorylation following phosphorylation at Thr197. Using these two substrates and mammalian-expressed PDK-1, we can elucidate a possible two-step process for the activation of the C-subunit: initial phosphorylation on the activation loop at Thr197 by PDK-1, or a PDK-1-like enzyme, followed by second cis autophosphorylation step at Ser338.     

Effects of Protein Kinase Inhibitors on Morphology and Function of Cultured Bovine Adrenal Chromaffin Cells: KN-62 Inhibits Secretory Function by Blocking Stimulated Ca2+ Entry

Abstract: In cultured bovine adrenal chromaffin cells, a nonselective protein kinase inhibitor, staurosporine, inhibits secretory function and induces neurite outgrowth. In the present study, effects of other nonselective protein kinase inhibitors (K-252a, H-7, and H-8) and reportedly selective protein kinase inhibitors (KN-62 and chelerythrine chloride) were examined on bovine adrenal chromaffin cell morphology, secretory function, and ⁴⁵Ca²⁺ uptake. Treatment of chromaffin cells with 10 µ M K-252a, 50 µ M H-7, or 50 µ M H-8 induced changes in cell morphology within 3 h; these compounds also induced a time-dependent inhibition of stimulated catecholamine release. Chelerythrine chloride, a selective inhibitor of Ca²⁺/phospholipid-dependent protein kinase, did not induce outgrowth or inhibit secretory function under our treatment conditions. KN-62, a selective inhibitor of Ca²⁺/calmodulin-dependent protein kinase II (CaMK II), significantly inhibited stimulated catecholamine release (IC₅₀ value of 0.32 µ M ), but had no effect on cell morphology. The reduction of secretory function induced by 1 µ M KN-62 was significant within 5 min and rapidly reversible. Unlike H-7, H-8, and staurosporine, KN-62 significantly inhibited stimulated ⁴⁵Ca²⁺ uptake. KN-04, a structural analogue of KN-62 that does not inhibit CaMK II, inhibited stimulated ⁴⁵Ca²⁺ uptake and catecholamine release like KN-62. These studies indicate that KN-62 inhibits secretory function via the direct blockade of activated Ca²⁺ influx. The nonselective inhibitors, K-252a, H-7, H-8, and staurosporine, inhibit secretory function by another mechanism, perhaps one involving alterations in the cytoskeleton.     

Structural insights into how irreversible inhibitors can overcome drug resistance in EGFR

Resistance to kinase-targeted cancer drugs has recently been linked to a single point mutation in the ATP binding site of the kinase. In EGFR, the crucial Thr790 gatekeeper residue is mutated to a Met and prevents reversible ATP competitive inhibitors from binding. Irreversible 4-(phenylamino)quinazolines have been shown to overcome this drug resistance and are currently in clinical trials. In order to obtain a detailed structural understanding of how irreversible inhibitors overcome drug resistance, we used Src kinase as a model system for drug resistant EGFR-T790M. We report the first crystal structure of a drug resistant kinase in complex with an irreversible inhibitor. This 4-(phenylamino)quinazoline inhibits wild type and drug resistant EGFR in vitro at low nM concentrations. The co-crystal structure of drug resistant cSrc-T338M kinase domain provides the structural basis of this activity.     

Structural and mechanistic underpinnings of the differential drug sensitivity of EGFR mutations in non-small cell lung cancer

EGFR and other ErbB-family tyrosine kinases are overexpressed in many human tumors, and their aberrant expression and mutational activation is associated with the development, progression and aggressiveness of a number of malignancies. Thus the EGFR kinase has long been recognized as a potential drug target in oncology, and small-molecule inhibitors have been under development for more than two decades. As a result of their effectiveness in treating non-small cell lung cancers (NSCLCs) driven by somatic mutations in the EGFR kinase, gefitinib and erlotinib were the first EGFR tyrosine kinase inhibitors (TKIs) approved for clinical use. Ironically, these drugs found their target against mutant forms of the EGFR kinase, which have altered enzyme active sites, and not against the wild type (WT) kinase against which their potency and selectivity was carefully honed. Here we review recent structural and enzymological studies that explore the exquisite sensitivity of a subset of these lung cancer mutants to gefitinib and erlotinib. We discuss available structural evidence for the mechanisms of activation of the EGFR kinase by these mutants, and compare it to physiologic activation of the kinase by ligand-induced dimerization. Finally, we consider the mechanisms by which the secondary T790M “gatekeeper” mutation confers resistance to gefitinib and erlotinib.     

Phosphorylation of Mutationally Introduced Tyrosine in the Activation Loop of HER2 Confers Gain-of-Function Activity

Amplification, overexpression, and somatic mutation of the HER2 gene have been reported to play a critical role in tumorigenesis of various cancers. The HER2 H878Y mutation was recently reported in 11% of hepatocellular carcinoma (HCC) patients. However, its functional impact on the HER2 protein and its role in tumorigenesis has not been determined. Here, we show that HER2 H878Y is a gain-of-function mutation. Y878 represents a phosphorylation site, and phospho-Y878 interacts with R898 residue to stabilize the active conformation of HER2, thereby enhancing its kinase activity. H878Y mutant is transforming and the transformed cells are sensitive to HER2 kinase inhibitors. Thus, our study reveals the following novel mechanism underlying the tumorigenic function of the HER2 H878Y mutation: the introduction of a tyrosine residue into the kinase activation loop via mutagenesis modulates the conformation of the kinase, thereby enhancing its activity.     

Molecular mechanisms and design principles for promiscuous inhibitors to avoid drug resistance: Lessons learned from HIV‐1 protease inhibition

Molecular recognition is central to biology and ranges from highly selective to broadly promiscuous. The ability to modulate specificity at will is particularly important for drug development, and discovery of mechanisms contributing to binding specificity is crucial for our basic understanding of biology and for applications in health care. In this study, we used computational molecular design to create a large dataset of diverse small molecules with a range of binding specificities. We then performed structural, energetic, and statistical analysis on the dataset to study molecular mechanisms of achieving specificity goals. The work was done in the context of HIV‐1 protease inhibition and the molecular designs targeted a panel of wild‐type and drug‐resistant mutant HIV‐1 protease structures. The analysis focused on mechanisms for promiscuous binding to bind robustly even to resistance mutants. Broadly binding inhibitors tended to be smaller in size, more flexible in chemical structure, and more hydrophobic in nature compared to highly selective ones. Furthermore, structural and energetic analyses illustrated mechanisms by which flexible inhibitors achieved binding; we found ligand conformational adaptation near mutation sites and structural plasticity in targets through torsional flips of asymmetric functional groups to form alternative, compensatory packing interactions or hydrogen bonds. As no inhibitor bound to all variants, we designed small cocktails of inhibitors to do so and discovered that they often jointly covered the target set through mechanistic complementarity. Furthermore, using structural plasticity observed in experiments, and potentially in simulations, is suggested to be a viable means of designing adaptive inhibitors that are promiscuous binders. Proteins 2015; 83:351–372. © 2014 Wiley Periodicals, Inc.     

Targeting transcription of MCL-1 sensitizes HER2-amplified breast cancers to HER2 inhibitors

Human epidermal growth factor receptor 2 gene (HER2) is focally amplified in approximately 20% of breast cancers. HER2 inhibitors alone are not effective, and sensitizing agents will be necessary to move away from a reliance on heavily toxic chemotherapeutics. We recently demonstrated that the efficacy of HER2 inhibitors is mitigated by uniformly low levels of the myeloid cell leukemia 1 (MCL-1) endogenous inhibitor, NOXA. Emerging clinical data have demonstrated that clinically advanced cyclin-dependent kinase (CDK) inhibitors are effective MCL-1 inhibitors in patients, and, importantly, well tolerated. We, therefore, tested whether the CDK inhibitor, dinaciclib, could block MCL-1 in preclinical HER2-amplified breast cancer models and therefore sensitize these cancers to dual HER2/EGFR inhibitors neratinib and lapatinib, as well as to the novel selective HER2 inhibitor tucatinib. Indeed, we found dinaciclib suppresses MCL-1 RNA and is highly effective at sensitizing HER2 inhibitors both in vitro and in vivo. This combination was tolerable in vivo. Mechanistically, liberating the effector BCL-2 protein, BAK, from MCL-1 results in robust apoptosis. Thus, clinically advanced CDK inhibitors may effectively combine with HER2 inhibitors and present a chemotherapy-free therapeutic strategy in HER2-amplified breast cancer, which can be tested immediately in the clinic.Subject terms: Breast cancer, Target identification     

Structural basis for specificity of TGFβ family receptor small molecule inhibitors

Transforming growth factor-β (TGFβ) receptor kinase inhibitors have a great therapeutic potential. SB431542 is one of the mainly used kinase inhibitors of the TGFβ/Activin pathway receptors, but needs improvement of its EC₅₀ (EC₅₀ = 1 μM) to be translated to clinical use. A key feature of SB431542 is that it specifically targets receptors from the TGFβ/Activin pathway but not the closely related receptors from the bone morphogenic proteins (BMP) pathway. To understand the mechanisms of this selectivity, we solved the crystal structure of the TGFβ type I receptor (TβRI) kinase domain in complex with SB431542. We mutated TβRI residues coordinating SB431542 to their counterparts in activin-receptor like kinase 2 (ALK2), a BMP receptor kinase, and tested the kinase activity of mutated TβRI. We discovered that a Ser280Thr mutation yielded a TβRI variant that was resistant to SB431542 inhibition. Furthermore, the corresponding Thr283Ser mutation in ALK2 yielded a BMP receptor sensitive to SB431542. This demonstrated that Ser280 is the key determinant of selectivity for SB431542. This work provides a framework for optimising the SB431542 scaffold to more potent and selective inhibitors of the TGFβ/Activin pathway.     

Structural analysis of the lymphocyte-specific kinase Lck in complex with non-selective and Src family selective kinase inhibitors.

BACKGROUND: The lymphocyte-specific kinase Lck is a member of the Src family of non-receptor tyrosine kinases. Lck catalyzes the initial phosphorylation of T-cell receptor components that is necessary for signal transduction and T-cell activation. On the basis of both biochemical and genetic studies, Lck is considered an attractive cell-specific target for the design of novel T-cell immunosuppressants. To date, the lack of detailed structural information on the mode of inhibitor binding to Lck has limited the discovery of novel Lck inhibitors. RESULTS: We report here the high-resolution crystal structures of an activated Lck kinase domain in complex with three structurally distinct ATP-competitive inhibitors: AMP-PNP (a non-selective, non-hydrolyzable ATP analog); staurosporine (a potent but non-selective protein kinase inhibitor); and PP2 (a potent Src family selective protein tyrosine kinase inhibitor). Comparison of these structures reveals subtle but important structural changes at the ATP-binding site. Furthermore, PP2 is found to access a deep, hydrophobic pocket near the ATP-binding cleft of the enzyme; this binding pocket is not occupied by either AMP-PNP or staurosporine. CONCLUSIONS: The potency of staurosporine against Lck derives in part from an induced movement of the glycine-rich loop of the enzyme upon binding of this ligand, which maximizes the van der Waals interactions present in the complex. In contrast, PP2 binds tightly and selectively to Lck and other Src family kinases by making additional contacts in a deep, hydrophobic pocket adjacent to the ATP-binding site; the amino acid composition of this pocket is unique to Src family kinases. The structures of these Lck complexes offer useful structural insights as they demonstrate that kinase selectivity can be achieved with small-molecule inhibitors that exploit subtle topological differences among protein kinases.     

Potent selective inhibitors of protein kinase C

A series of potent, selective inhibitors of protein kinase C has been derived from the structural lead provided by the microbial broth products, staurosporine and K252a. Our inhibitors block PCK in intact cells (platelets and T cells), and prevent the proliferation of mononuclear cells in response to interleukin 2 (IL2).     

Isoliquiritigenin Induces Apoptosis and Inhibits Xenograft Tumor Growth of Human Lung Cancer Cells by Targeting Both Wild Type and L858R/T790M Mutant EGFR

Non-small-cell lung cancer (NSCLC) is associated with diverse genetic alterations including mutation of epidermal growth factor receptor (EGFR). Isoliquiritigenin (ILQ), a chalcone derivative, possesses anticancer activities. In the present study, we investigated the effects of ILQ on the growth of tyrosine kinase inhibitor (TKI)-sensitive and -resistant NSCLC cells and elucidated its underlying mechanisms. Treatment with ILQ inhibited growth and induced apoptosis in both TKI-sensitive and -resistant NSCLC cells. ILQ-induced apoptosis was associated with the cleavage of caspase-3 and poly-(ADP-ribose)-polymerase, increased expression of Bim, and reduced expression of Bcl-2. In vitro kinase assay results revealed that ILQ inhibited the catalytic activity of both wild type and double mutant (L858R/T790M) EGFR. Treatment with ILQ inhibited the anchorage-independent growth of NIH3T3 cells stably transfected with either wild type or double-mutant EGFR with or without EGF stimulation. ILQ also reduced the phosphorylation of Akt and ERK1/2 in both TKI-sensitive and -resistant NSCLC cells, and attenuated the kinase activity of Akt1 and ERK2 in vitro. ILQ directly interacted with both wild type and double-mutant EGFR in an ATP-competitive manner. A docking model study showed that ILQ formed two hydrogen bonds (Glu-762 and Met-793) with wild type EGFR and three hydrogen bonds (Lys-745, Met-793, and Asp-855) with mutant EGFR. ILQ attenuated the xenograft tumor growth of H1975 cells, which was associated with decreased expression of Ki-67 and diminished phosphorylation of Akt and ERK1/2. Taken together, ILQ suppresses NSCLC cell growth by directly targeting wild type or mutant EGFR.     

Comparison of the EGFR resistance mutation profiles generated by EGFR-targeted tyrosine kinase inhibitors and the impact of drug combinations

Recent clinical data indicates that the emergence of mutant drug-resistant kinase alleles may be particularly relevant for targeted kinase inhibitors. In order to explore how different classes of targeted therapies impact upon resistance mutations, we performed EGFR (epidermal-growth-factor receptor) resistance mutation screens with erlotinib, lapatinib and CI-1033. Distinct mutation spectra were generated with each inhibitor and were reflective of their respective mechanisms of action. Lapatinib yielded the widest variety of mutations, whereas mutational variability was lower in the erlotinib and CI-1033 screens. Lapatinib was uniquely sensitive to mutations of residues located deep within the selectivity pocket, whereas mutation of either Gly(796) or Cys(797) resulted in a dramatic loss of CI-1033 potency. The clinically observed T790M mutation was common to all inhibitors, but occurred with varying frequencies. Importantly, the presence of C797S with T790M in the same EGFR allele conferred complete resistance to erlotinib, lapatinib and CI-1033. The combination of erlotinib and CI-1033 effectively reduced the number of drug-resistant clones, suggesting a possible clinical strategy to overcome drug resistance. Interestingly, our results also indicate that co-expression of ErbB2 (v-erb-b2 erythroblastic leukaemia viral oncogene homologue 2) has an impact upon the EGFR resistance mutations obtained, suggesting that ErbB2 may play an active role in the acquisition of drug-resistant mutations.     

Completing the structural family portrait of the human EphB tyrosine kinase domains

The EphB receptors have key roles in cell morphology, adhesion, migration and invasion, and their aberrant action has been linked with the development and progression of many different tumor types. Their conflicting expression patterns in cancer tissues, combined with their high sequence and structural identity, present interesting challenges to those seeking to develop selective therapeutic molecules targeting this large receptor family. Here, we present the first structure of the EphB1 tyrosine kinase domain determined by X‐ray crystallography to 2.5Å. Our comparative crystalisation analysis of the human EphB family kinases has also yielded new crystal forms of the human EphB2 and EphB4 catalytic domains. Unable to crystallize the wild‐type EphB3 kinase domain, we used rational engineering (based on our new structures of EphB1, EphB2, and EphB4) to identify a single point mutation which facilitated its crystallization and structure determination to 2.2 Å. This mutation also improved the soluble recombinant yield of this kinase within Escherichia coli, and increased both its intrinsic stability and catalytic turnover, without affecting its ligand‐binding profile. The partial ordering of the activation loop in the EphB3 structure alludes to a potential cis‐phosphorylation mechanism for the EphB kinases. With the kinase domain structures of all four catalytically competent human EphB receptors now determined, a picture begins to emerge of possible opportunities to produce EphB isozyme‐selective kinase inhibitors for mechanistic studies and therapeutic applications.