Clinical stage EGFR inhibitors irreversibly alkylate Bmx kinase

Irreversible HER/erbB inhibitors selectively inhibit HER-family kinases by targeting a unique cysteine residue located within the ATP-binding pocket. Sequence alignment reveals that this rare cysteine is also present in ten other protein kinases including all five Tec-family members. We demonstrate that the Tec-family kinase Bmx is potently inhibited by irreversible modification at Cys496 by clinical stage EGFR inhibitors such as CI-1033. This cross-reactivity may have significant clinical implications.     

A chemical genetic approach for covalent inhibition of analogue-sensitive aurora kinase

The perturbation of protein kinases with small organic molecules is a powerful approach to dissect kinase function in complex biological systems. Covalent kinase inhibitors that target thiols in the ATP binding pocket of the kinase domain proved to be ideal reagents for the investigation of highly dynamic cellular processes. However, due to the covalent inhibitors' possible off-target reactivities, it is required that the overall shape of the inhibitor as well as the intrinsic reactivity of the electrophile are precisely tuned to favor the reaction with only the desired cysteine. Here we report on the design and biological characterization of covalent anilinoquinazolines as potent inhibitors of genetically engineered Aurora kinase in fission yeast.     

Discovery of potent,highly selective covalent irreversible BTK inhibitors from a fragment hit

Bruton's Tyrosine Kinase (BTK) is a member of the TEC kinase family that is expressed in cells of hematopoietic lineage (e.g., in B cells, macrophages, monocytes, and mast cells). Small molecule covalent irreversible BTK inhibitor targeting Cys481 within the ATP-binding pocket, for example ibrutinib, has been applied in the treatment of B-cell malignancies. Starting from a fragment hit, we discovered a novel series of potent covalent irreversible BTK inhibitors that occupy selectivity pocket of the active site of the BTK kinase domain. Guided by X-ray structures and a fragment-based drug design (FBDD) approach, we generated molecules showing comparable cellular potency to ibrutinib and higher kinome selectivity against undesirable off-targets like EGFR.     

Mutation of threonine 766 in the epidermal growth factor receptor reveals a hotspot for resistance formation against selective tyrosine kinase inhibitors

Small molecule inhibitors of protein tyrosine kinases such as STI571 represent a major new class of therapeutics for target-selective treatment of human cancer. Clinical resistance formation to the BCR-ABL inhibitor STI571 has been observed in patients with advanced chronic myeloid leukemia and was frequently caused by a C to T single nucleotide change in the Abl kinase domain, which substituted Thr-315 with isoleucine and rendered BCR-ABL resistant to STI571 inhibition. The corresponding mutation in the epidermal growth factor receptor (EGFR) tyrosine kinase replaced Thr-766 of the EGFR by methionine and dramatically reduced the sensitivity of EGFR to inhibition by selective 4-anilinoquinazoline inhibitors such as PD153035. Inhibitor-resistant EGFR exhibited the same signaling capacity as wild-type receptor in vivo and provides a useful tool for analyzing EGFR-mediated signal transduction. Our data identify Thr-766 of the EGFR as a structural determinant that bears the potential to become a relevant feature in resistance formation during cancer therapy with EGFR-specific 4-anilinoquinazoline inhibitors.     

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.     

Long-lasting inhibition of EGFR autophosphorylation in A549 tumor cells by intracellular accumulation of non-covalent inhibitors

In the present study, a small set of reversible or irreversible 4-anilinoquinazoline EGFR inhibitors was tested in A549 cells at early (1 h) and late (8 h) time points after inhibitor removal from culture medium. A combination of assays was employed to explain the observed long-lasting inhibition of EGFR autophosphorylation. We found that EGFR inhibition at 8 h can be due, besides to the covalent interaction of the inhibitor with Cys797, as for PD168393 (2) and its prodrug 4, to the intracellular accumulation of non-covalent inhibitors by means of an active cell uptake, as for 5 and 6. Compounds 5–6 showed similar potency and duration of inhibition of EGFR autophosphorylation as the covalent inhibitor 2, while being devoid of reactive groups forming covalent bonds with protein thiols.     

c-Src trafficking and co-localization with the EGF receptor promotes EGF ligand-independent EGF receptor activation and signaling

c-Src is a non-receptor tyrosine kinase that associates with both the plasma membrane and endosomal compartments. In many human cancers, especially breast cancer, c-Src and the EGF receptor (EGFR) are overexpressed. Dual overexpression of c-Src and EGFR correlates with a Src-dependent increase in activation of EGFR, and synergism between these two tyrosine kinases increases the mitogenic activity of EGFR. Despite extensive studies of the functional interaction between c-Src and EGFR, little is known about the interactions in the trafficking pathways for the two proteins and how that influences signaling. Given the synergism between c-Src and EGFR, and the finding that EGFR is internalized and can signal from endosomes, we hypothesized that c-Src and EGFR traffic together through the endocytic pathway. Here we use a regulatable c-SrcGFP fusion protein that is a bona fide marker for c-Src to show that c-Src undergoes constitutive macropinocytosis from the plasma membrane into endocytic compartments. The movement of c-Src was dependent on its tyrosine kinase activity. Stimulation of cells with EGF revealed that c-Src traffics into the cell with activated EGFR and that c-Src expression and kinase activity prolongs EGFR activation. Surprisingly, even in the absence of EGF addition, c-Src expression induced activation of EGFR and of EGFR-mediated downstream signaling targets ERK and Shc. These data suggest that the synergy between c-Src and EGFR also occurs as these two kinases traffic together, and that their co-localization promotes EGFR-mediated signaling.     

The discovery of highly selective erbB2 (Her2) inhibitors for the treatment of cancer

The synthesis and biological evaluation of potent and selective inhibitors of the erbB2 kinase is presented. Based on the 4-anilinoquinazoline chemotype, the syntheses of several new series of erbB2 inhibitors are described with quinazoline and pyrido[4,3-d]pyrimidine cores. The vast majority of these compounds are found to be >100x selective over the closely related EGFR kinase. Two lead compounds are further shown to have low clearance and moderate bioavailability in rat.     

Structure-based design and synthesis of 1H-pyrazolo[3,4-d]pyrimidin-4-amino derivatives as Janus kinase 3 inhibitors

Janus kinases (JAKs) regulate various inflammatory and immune responses and are targets for the treatment of inflammatory and immune diseases. Here we report the discovery and optimization of 1H-pyrazolo[3,4-d]pyrimidin-4-amino as covalent JAK3 inhibitors that exploit a unique cysteine (Cys909) residue in JAK3. Our optimization study gave compound 12a, which exhibited potent JAK3 inhibitory activity (IC₅₀ of 6.2?nM) as well as excellent JAK kinase selectivity (>60-fold). In cellular assay, 12a exhibited potent immunomodulating effect on IL-2-stimulated T cell proliferation (IC₅₀ of 9.4?μM). Further, compound 12a showed efficacy in delayed hypersensitivity assay. The data supports the further investigation of these compounds as novel JAKs inhibitors.     

Src mediates prolactin-dependent proliferation of T47D and MCF7 cells via the activation of focal adhesion kinase/Erk1/2 and phosphatidylinositol 3-kinase pathways

Prolactin (PRL) stimulates breast cancer cell proliferation; however, the involvement of PRL-activated signaling molecules in cell proliferation is not fully established. Here we studied the role of c-Src on PRL-stimulated proliferation of T47D and MCF7 breast cancer cells. We initially observed that PRL-dependent activation of focal adhesion kinase (Fak), Erk1/2, and cell proliferation was mediated by c-Src in T47D cells, because expression of a dominant-negative form of c-Src (SrcDM, K295A/Y527F) blocked the PRL-dependent effects. The Src inhibitor PP1 abrogated PRL-dependent in vivo activation of Fak, Erk1/2, p70S6K, and Akt and the proliferation of T47D and MCF7 cells; Janus kinase 2 (Jak2) activation was not affected. However, in vitro, Fak and Jak2 kinases were not directly inhibited by PP1, demonstrating the effect of PP1 on c-Src kinase as an upstream activator of Fak. Expression of Fak mutant Y397F abrogated PRL-dependent activation of Fak, Erk1/2, and thymidine incorporation, but had no effect on p70S6K and Akt kinases. MAPK kinase 1/2 (Mek1/2) inhibitor PD184352 blocked PRL-induced stimulation of Erk1/2 and cell proliferation; however, p70S6K and Akt activation were unaffected. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 abolished cell proliferation and activation of p70S6K and Akt; however, PRL-dependent activation of Erk1/2 was not modified. Moreover, we show that both c-Src/PI3K and c-Src/Fak/Erk1/2 pathways are involved in the up-regulation of c-myc and cyclin d1 expression mediated by PRL. The previous findings suggest the existence of two PRL-dependent signaling cascades, initiated by the c-Src-mediated activation of Fak/Erk1/2 and PI3K pathways that, subsequently, control the expression of c-Myc and cyclin D1 and the proliferation of T47D and MCF7 breast cancer cells.     

Discovery of novel substituted benzo-anellated 4-benzylamino pyrrolopyrimidines as dual EGFR and VEGFR2 inhibitors

The quinazoline scaffold is the main part of many marketed EGFR inhibitors. Resistance developments against those inhibitors enforced the search for novel structural lead compounds. We developed novel benzo-anellated 4-benzylamine pyrrolopyrimidines with varied substitution patterns at both the molecular scaffold and the attached residue in the 4-position. The structure-dependent affinities towards EGFR are discussed and first nanomolar derivatives have been identified. Docking studies were carried out for EGFR in order to explore the potential binding mode of the novel inhibitors. As the receptor tyrosine kinase VEGFR2 recently gained an increasing interest as an upregulated signaling kinase in many solid tumors and in tumor metastasis we determined the affinity of our compounds to inhibit VEGFR2. So we identified novel dually acting EGFR and VEGFR2 inhibitors for which first anticancer screening data are reported. Those data indicate a stronger antiproliferative effect of a VEGFR2 inhibition compared to the EGFR inhibition.     

AST1306, a novel irreversible inhibitor of the epidermal growth factor receptor 1 and 2, exhibits antitumor activity both in vitro and in vivo

Despite the initial response to the reversible, ATP-competitive quinazoline inhibitors that target ErbB-family, such a subset of cancer patients almost invariably develop resistance. Recent studies have provided compelling evidence that irreversible ErbB inhibitors have the potential to override this resistance. Here, we found that AST1306, a novel anilino-quinazoline compound, inhibited the enzymatic activities of wild-type epidermal growth factor receptor (EGFR) and ErbB2 as well as EGFR resistant mutant in both cell-free and cell-based systems. Importantly, AST1306 functions as an irreversible inhibitor, most likely through covalent interaction with Cys797 and Cys805 in the catalytic domains of EGFR and ErbB2, respectively. Further studies showed that AST1306 inactivated pathways downstream of these receptors and thereby inhibited the proliferation of a panel of cancer cell lines. Although the activities of EGFR and ErbB2 were similarly sensitive to AST1306, ErbB2-overexpressing cell lines consistently exhibited more sensitivity to AST1306 antiproliferative effects. Consistent with this, knockdown of ErbB2, but not EGFR, decreased the sensitivity of SK-OV-3 cells to AST1306. In vivo, AST1306 potently suppressed tumor growth in ErbB2-overexpressing adenocarcinoma xenograft and FVB-2/N(neu) transgenic breast cancer mouse models, but weakly inhibited the growth of EGFR-overexpressing tumor xenografts. Tumor growth inhibition induced by a single dose of AST1306 in the SK-OV-3 xenograft model was accompanied by a rapid (within 2 h) and sustained (≥24 h) inhibition of both EGFR and ErbB2, consistent with an irreversible inhibition mechanism. Taken together, these results establish AST1306 as a selective, irreversible ErbB2 and EGFR inhibitor whose growth-inhibitory effects are more potent in ErbB2-overexpressing cells.     

Peroxide-dependent sulfenylation of the EGFR catalytic site enhances kinase activity

Protein sulfenylation is a post-translational modification of emerging importance in higher eukaryotes. However, investigation of its diverse roles remains challenging, particularly within a native cellular environment. Herein we report the development and application of DYn-2, a new chemoselective probe for detecting sulfenylated proteins in human cells. These studies show that epidermal growth factor receptor-mediated signaling results in H(2)O(2) production and oxidation of downstream proteins. In addition, we demonstrate that DYn-2 has the ability to detect differences in sulfenylation rates within the cell, which are associated with differences in target protein localization. We also show that the direct modification of epidermal growth factor receptor by H(2)O(2) at a critical active site cysteine (Cys797) enhances its tyrosine kinase activity. Collectively, our findings reveal sulfenylation as a global signaling mechanism that is akin to phosphorylation and has regulatory implications for other receptor tyrosine kinases and irreversible inhibitors that target oxidant-sensitive cysteines in proteins.     

3D-QSAR studies on c-Src kinase inhibitors and docking analyses of a potent dual kinase inhibitor of c-Src and c-Abl kinases

Three-dimensional quantitative structure-activity relationship (3D-QSAR) analyses were carried out on quinazoline, quinoline, and cyanoquinoline derivatives inhibiting c-Src kinase. Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) 3D-QSAR models were developed. The conventional r2 values for CoMFA and CoMSIA are 0.93 and 0.89, respectively. In addition, a homology model of c-Src kinase with the activation loop resembling the active conformation was constructed using the crystal structure of the kinase domain of Lck. The ATP binding pocket of the active form of c-Src is similar to that of the c-Abl kinase in which the activation loop resembles that of an active form. One of the potent c-Src and c-Abl dual kinase inhibitors (77 or SKI-606) was docked inside the active sites of both c-Src and c-Abl. The orientation and hydrogen bonding interactions of 77 are similar in both kinases. The results of 3D-QSAR analyses and structure based studies will be useful for the design of novel c-Src and c-Abl dual kinase inhibitors.     

TRANCE, a TNF family member, activates Akt/PKB through a signaling complex involving TRAF6 and c-Src

TRANCE, a TNF family member, and its receptor, TRANCE-R, are critical regulators of dendritic cell and osteoclast function. Here, we demonstrate that TRANCE activates the antiapoptotic serine/threonine kinase Akt/PKB through a signaling complex involving c-Src and TRAF6. A deficiency in c-Src or addition of Src family kinase inhibitors blocks TRANCE-mediated PKB activation in osteoclasts. c-Src and TRAF6 interact with each other and with TRANCE-R upon receptor engagement. TRAF6, in turn, enhances the kinase activity of c-Src leading to tyrosine phosphorylation of downstream signaling molecules such as c-Cbl. These results define a mechanism by which TRANCE activates Src family kinases and PKB and provide evidence of cross-talk between TRAF proteins and Src family kinases.     

Discovery of new quinazoline derivatives as irreversible dual EGFR/HER2 inhibitors and their anticancer activities – Part 1

Overexpression of EGFR and HER2 are observed in many breast, ovarian, colon and prostate cancers. The second and third generation irreversible EGFR/HER2 dual kinase inhibitors became popular after the approval of Afatinib by FDA to overcome the mutation related problem. To find efficacious drug candidates, a series of novel quinazoline derivatives were designed, synthesized and evaluated as dual EGFR/HER2 tyrosine kinase (TK) inhibitors. Selected twenty four compounds were reported here with significant inhibitory activities against EGFR/HER2 tyrosine kinases. Several compounds showed nanomolar IC₅₀ values. In vitro studies of quinazoline derivatives were done on NCI-H1975, HCC827, A431, MDA MB-453 cell lines. The compounds 1a, 1d and 1v were found more potent compared to standard drug afatinib. In vivo efficacy study of 1d on nude mice NCI-H1975 tumour xenograft model was discussed.     

The beta(2)-adrenergic receptor mediates extracellular signal-regulated kinase activation via assembly of a multi-receptor complex with the epidermal growth factor receptor

Many G protein-coupled receptors (GPCRs) activate MAP kinases by stimulating tyrosine kinase signaling cascades. In some systems, GPCRs stimulate tyrosine phosphorylation by inducing the "transactivation" of a receptor tyrosine kinase (RTK). The mechanisms underlying GPCR-induced RTK transactivation have not been clearly defined. Here we report that GPCR activation mimics growth factor-mediated stimulation of the epidermal growth factor receptor (EGFR) with respect to many facets of RTK function. beta(2)-Adrenergic receptor (beta(2)AR) stimulation of COS-7 cells induces EGFR dimerization, tyrosine autophosphorylation, and EGFR internalization. Coincident with EGFR transactivation, isoproterenol exposure induces the formation of a multireceptor complex containing both the beta(2)AR and the "transactivated" EGFR. beta(2)AR-mediated EGFR phosphorylation and subsequent beta(2)AR stimulation of extracellular signal-regulated kinase (ERK) 1/2 are sensitive to selective inhibitors of both EGFR and Src kinases, indicating that both kinases are required for EGFR transactivation. beta(2)AR-dependent signaling to ERK1/2, like direct EGF stimulation of ERK1/2 activity, is sensitive to inhibitors of clathrin-mediated endocytosis, suggesting that signaling downstream of both the EGF-activated and the GPCR-transactivated EGFRs requires a productive engagement of the complex with the cellular endocytic machinery. Thus, RTK transactivation is revealed to be a process involving both association of receptors of distinct classes and the interaction of the transactivated RTK with the cells endocytic machinery.     

NADPH-oxidase-dependent reactive oxygen species mediate EGFR transactivation by FPRL1 in WKYMVm-stimulated human lung cancer cells

Cross talk between unrelated cell surface receptors, such as G-protein-coupled receptors (GPCR) and receptor tyrosine kinases (RTK), is a crucial signaling mechanism to expand the cellular communication network. We investigated the ability of the GPCR formyl peptide receptor-like 1 (FPRL1) to transactivate the RTK epidermal growth factor receptor (EGFR) in CaLu-6 cells. We observed that stimulation with WKYMVm, an FPRL1 agonist isolated by screening synthetic peptide libraries, induces EGFR tyrosine phosphorylation, p47^phox phosphorylation, NADPH-oxidase-dependent superoxide generation, and c-Src kinase activity. As a result of EGFR transactivation, phosphotyrosine residues provide docking sites for recruitment and triggering of the STAT3 pathway. WKYMVm-induced EGFR transactivation is prevented by the FPRL1-selective antagonist WRWWWW, by pertussis toxin (PTX), and by the c-Src inhibitor PP2. The critical role of NADPH-oxidase-dependent superoxide generation in this cross-talk mechanism is corroborated by the finding that apocynin or a siRNA against p22^phox prevents EGFR transactivation and c-Src kinase activity. In addition, WKYMVm promotes CaLu-6 cell growth, which is prevented by PTX and by WRWWWW. These results highlight the role of FPRL1 as a potential target of new drugs and suggest that targeting both FPRL1 and EGFR may yield superior therapeutic effects compared with targeting either receptor separately.     

Synthesis and Characterization of Novel Quinazoline Type Inhibitors for Mutant and Wild-Type EGFR and RICK Kinases

The development of selective protein kinase inhibitors has become an important area of drug discovery for the treatment of different diseases. We report the synthesis and characterization of a series of novel quinazoline derivatives against three therapeutically important and pharmacologically related kinases: 1) epidermal growth factor receptor (EGFR; wild type and mutant) in the field of cancer, 2) receptor-interacting caspase-like apoptosis-regulatory kinase (RICK) in the field of inflammation, and 3) pUL97 of human cytomegalovirus (HCMV). For reference purpose we have synthesized the four clinically relevant quinazolines, including the lead compounds, which we previously identified for RICK and pUL97. A total of 52 quinazoline derivatives were synthesized and tested on the basis of these leads to specifically target the hydrophobic pocket of the ATP-binding site. Selected compounds were tested on wild-type and mutant forms of EGFR, RICK, and pUL97 kinases; their logP and logS values for assessing suitability as drugs were calculated and hit or lead compounds identified.