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Patients with non-small cell lung cancers that have kinase-activating epidermal growth factor receptor (EGFR) mutations are highly responsive to first- and second-generation EGFR inhibitors. However, these patients often relapse due to a secondary, drug-resistant mutation in EGFR whereby the gatekeeper threonine is converted to methionine (T790M). Several third-generation EGFR inhibitors have been developed that irreversibly inactivate T790M-EGFR while sparing wild-type EGFR, thus reducing epithelium-based toxicities. Using chemical proteomics, we show here that individual T790M-EGFR inhibitors exhibit strikingly distinct off-target profiles in human cells. The FDA-approved drug osimertinib (AZD9291), in particular, was found to covalently modify cathepsins in cell and animal models, which correlated with lysosomal accumulation of the drug. Our findings thus show how chemical proteomics can be used to differentiate covalent kinase inhibitors based on global selectivity profiles in living systems and identify specific off-targets of these inhibitors that may affect drug activity and safety., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Covalent inhibition is a reemerging paradigm in kinase drug design, but the roles of inhibitor binding affinity and chemical reactivity in overall potency are not well-understood. To characterize the underlying molecular processes at a microscopic level and determine the appropriate kinetic constants, specialized experimental design and advanced numerical integration of differential equations are developed. Previously uncharacterized investigational covalent drugs reported here are shown to be extremely effective epidermal growth factor receptor (EGFR) inhibitors (kinact/Ki in the range 10(5)-10(7) M(-1)s(-1)), despite their low specific reactivity (kinact ≤ 2.1 × 10(-3) s(-1)), which is compensated for by high binding affinities (Ki < 1 nM). For inhibitors relying on reactivity to achieve potency, noncovalent enzyme-inhibitor complex partitioning between inhibitor dissociation and bond formation is central. Interestingly, reversible binding affinity of EGFR covalent inhibitors is highly correlated with antitumor cell potency. Furthermore, cellular potency for a subset of covalent inhibitors can be accounted for solely through reversible interactions. One reversible interaction is between EGFR-Cys797 nucleophile and the inhibitor's reactive group, which may also contribute to drug resistance. Because covalent inhibitors target a cysteine residue, the effects of its oxidation on enzyme catalysis and inhibitor pharmacology are characterized. Oxidation of the EGFR cysteine nucleophile does not alter catalysis but has widely varied effects on inhibitor potency depending on the EGFR context (e.g., oncogenic mutations), type of oxidation (sulfinylation or glutathiolation), and inhibitor architecture. These methods, parameters, and insights provide a rational framework for assessing and designing effective covalent inhibitors.

The oncogenicity of the L858R mutant form of the epidermal growth factor receptor (EGFR) in non-small-cell lung cancer is thought to be due to the constitutive activation of its kinase domain. The selectivity of the marketed drugs gefitinib and erlotinib for L858R mutant is attributed to their specific recognition of the active kinase and to weaker ATP binding by L858R EGFR. We present crystal structures showing that neither L858R nor the drug-resistant L858R+T790M EGFR kinase domain is in the constitutively active conformation. Additional co-crystal structures show that gefitinib and dacomitinib, an irreversible anilinoquinazoline derivative currently in clinical development, may not be conformation specific for the active state of the enzyme. Structural data further reveal the potential mode of recognition of one of the autophosphorylation sites in the C-terminal tail, Tyr-1016, by the kinase domain. Biochemical and biophysical evidence suggest that the oncogenic mutations impact the conformational dynamics of the enzyme., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The title compound, C(14)H(13)BrN(2)O, was obtained by reaction of indan-1-yl methane-sulfonate with 2-amino-5-bromo-pyridin-3-ol in the presence of caesium carbonate. The indane ring system is approximately planar [all but one of the C atoms are coplanar within 0.03 Å, the latter atom being displaced by 0.206 (2) Å from the mean plane through the remaining atoms] and forms a dihedral angle of 58.41 (4)° with the pyridine ring. In the crystal, centrosymmetrically related mol-ecules are linked into dimers by N-H⋯N hydrogen bonds.

The title compound, C(12)H(13)ClN(6), was prepared by reaction of 4,5-dichloro-7H-pyrrolo[2,3-d]pyrimidine with 2-(1H-imid-azol-4-yl)-N-methyl-ethanamine, and the X-ray study confirmed that chloro-substituent in six-membered ring was replaced in the reaction. The exocyclic N atom environment is approximately coplanar with the pyrrolo[2,3-d]pyrimidine [corresponding dihedral angle is 5.5 (1)°], whereas the mean plane of the N-C-C-C link connecting with the imidazolyl ring is almost exactly orthogonal to the plane of the bicyclic system [dihedral angle = 91.6 (2)°]. The imidazolyl plane itself, however, forms a relatively small dihedral angle of 20.8 (1)° with the pyrrolo[2,3-d]pyrimidine plane. There are two independent N-H⋯N hydrogen bonds in the structure, which link mol-ecules into layers parallel to (03).

The reaction of (E)-tert-butyl 4-[3-(dimethyl-amino)acrylo-yl]piperidine-1-carboxyl-ate with methyl-hydrazine leads to the formation of the title compound, C(14)H(23)N(3)O(2), with a 1-methyl-1H-pyrazol-5-yl substituent. The plane of the pyrazole ring forms a dihedral angle of 33.4 (1)° with the approximate mirror plane of the piperidine ring.

Amplification and overexpression of erbB2 (Her-2/neu) proto-oncogene has been linked to human malignancies including tumors of the breast, ovary, and stomach. It has been implicated in tumor growth, sensitivity to standard chemotherapy, prognosis of patients, and disease-free survival. Although the clinical use of trastuzumab (Herceptin) has prolonged the survival of breast cancer patients with erbB2-overexpressing tumors, there is an urgent need for more potent and orally bioavailable small-molecule inhibitors. CP-724,714 is a potent inhibitor of erbB2 receptor autophosphorylation in intact cells and is currently undergoing phase I clinical trials. Here, we describe the effects of CP-724,714 in vitro and in vivo in human breast cancer models. CP-724,714 is selective for inhibiting growth of HER2-driven cell lines. In addition, we show that it induces G1 cell cycle block in erbB2-overexpressing BT-474 human breast carcinoma cells and inhibits erbB2 autophosphorylation in xenografts when administered p.o. to athymic mice. It induces a marked reduction of extracellular signal-regulated kinase and Akt phosphorylation, tumor cell apoptosis, and release of caspase-3. P.o. administration (q.d. or b.i.d.) of CP-724,714 inhibits the growth of erbB2-overexpressing tumors in athymic mice without overt adverse effects.

Bone is accrued and maintained primarily through the coupled actions of bone-forming osteoblasts and bone-resorbing osteoclasts. Cumulative in vitro studies indicated that proline-rich tyrosine kinase 2 (PYK2) is a positive mediator of osteoclast function and activity. However, our investigation of PYK2-/- mice did not reveal evidence supporting an essential function for PYK2 in osteoclasts either in vivo or in culture. We find that PYK2-/- mice have high bone mass resulting from an unexpected increase in bone formation. Consistent with the in vivo findings, mouse bone marrow cultures show that PYK2 deficiency enhances differentiation and activity of osteoprogenitor cells, as does expressing a PYK2-specific short hairpin RNA or dominantly interfering proteins in human mesenchymal stem cells. Furthermore, the daily administration of a small-molecule PYK2 inhibitor increases bone formation and protects against bone loss in ovariectomized rats, an established preclinical model of postmenopausal osteoporosis. In summary, we find that PYK2 regulates the differentiation of early osteoprogenitor cells across species and that inhibitors of the PYK2 have potential as a bone anabolic approach for the treatment of osteoporosis.

Focal adhesion kinase (FAK) is a member of a family of non-receptor protein-tyrosine kinases that regulates integrin and growth factor signaling pathways involved in cell migration, proliferation, and survival. FAK expression is increased in many cancers, including breast and prostate cancer. Here we describe perturbation of adhesion-mediated signaling with a FAK inhibitor, PF-573,228. In vitro, this compound inhibited purified recombinant catalytic fragment of FAK with an IC(50) of 4 nM. In cultured cells, PF-573,228 inhibited FAK phosphorylation on Tyr(397) with an IC(50) of 30-100 nM. Treatment of cells with concentrations of PF-573,228 that significantly decreased FAK Tyr(397) phosphorylation failed to inhibit cell growth or induce apoptosis. In contrast, treatment with PF-573,228 inhibited both chemotactic and haptotactic migration concomitant with the inhibition of focal adhesion turnover. These studies show that PF-573,228 serves as a useful tool to dissect the functions of FAK in integrin-dependent signaling pathways in normal and cancer cells and forms the basis for the generation of compounds amenable for preclinical and patient trials.

CP-673,451 is a potent inhibitor of platelet-derived growth factor beta-receptor (PDGFR-beta) kinase- and PDGF-BB-stimulated autophosphorylation of PDGFR-beta in cells (IC(50) = 1 nmol/L) being more than 450-fold selective for PDGFR-beta versus other angiogenic receptors (e.g., vascular endothelial growth factor receptor 2, TIE-2, and fibroblast growth factor receptor 2). Multiple models have been used to evaluate in vivo activity of CP-673,451 and to understand the pharmacology of PDGFR-beta inhibition and the effect on tumor growth. These models include an ex vivo measure of PDGFR-beta phosphorylation in glioblastoma tumors, a sponge model to measure inhibition of angiogenesis, and multiple models of tumor growth inhibition. Inhibition of PDGFR-beta phosphorylation in tumors correlates with plasma and tumor levels of CP-673,451. A dose of 33 mg/kg was adequate to provide >50% inhibition of receptor for 4 hours corresponding to an EC(50) of 120 ng/mL in plasma at C(max). In a sponge angiogenesis model, CP-673,451 inhibited 70% of PDGF-BB-stimulated angiogenesis at a dose of 3 mg/kg (q.d. x 5, p.o., corresponding to 5.5 ng/mL at C(max)). The compound did not inhibit vascular endothelial growth factor- or basic fibroblast growth factor-induced angiogenesis at concentrations which inhibited tumor growth. The antitumor efficacy of CP-673,451 was evaluated in a number of human tumor xenografts grown s.c. in athymic mice, including H460 human lung carcinoma, Colo205 and LS174T human colon carcinomas, and U87MG human glioblastoma multiforme. Once-daily p.o. x 10 days dosing routinely inhibited tumor growth (ED(50) < or = 33 mg/kg). These data show that CP-673,451 is a pharmacologically selective PDGFR inhibitor, inhibits tumor PDGFR-beta phosphorylation, selectively inhibits PDGF-BB-stimulated angiogenesis in vivo, and causes significant tumor growth inhibition in multiple human xenograft models.

The chemokines CCL3 and CCL5, as well as their shared receptor CCR1, are believed to play a role in the pathogenesis of several inflammatory diseases including rheumatoid arthritis, multiple sclerosis, and transplant rejection. In this study we describe the pharmacological properties of a novel small molecular weight CCR1 antagonist, CP-481,715 (quinoxaline-2-carboxylic acid [4(R)-carbamoyl-1(S)-(3-fluorobenzyl)-2(S),7-dihydroxy-7-methyloctyl]amide). Radiolabeled binding studies indicate that CP-481,715 binds to human CCR1 with a Kd of 9.2 nm and displaces 125I-labeled CCL3 from CCR1-transfected cells with an IC50 of 74 nm. CP-481,715 lacks intrinsic agonist activity but fully blocks the ability of CCL3 and CCL5 to stimulate receptor signaling (guanosine 5'-O-(thiotriphosphate) incorporation; IC50 = 210 nm), calcium mobilization (IC50 = 71 nm), monocyte chemotaxis (IC50 = 55 nm), and matrix metalloproteinase 9 release (IC50 = 54 nm). CP-481,715 retains activity in human whole blood, inhibiting CCL3-induced CD11b up-regulation and actin polymerization (IC50 = 165 and 57 nm, respectively) on monocytes. Furthermore, it behaves as a competitive and reversible antagonist. CP-481,715 is >100-fold selective for CCR1 as compared with a panel of G-protein-coupled receptors including related chemokine receptors. Evidence for its potential use in human disease is suggested by its ability to inhibit 90% of the monocyte chemotactic activity present in 11/15 rheumatoid arthritis synovial fluid samples. These data illustrate that CP-481,715 is a potent and selective antagonist for CCR1 with therapeutic potential for rheumatoid arthritis and other inflammatory diseases.

Aberrant expression or activity of epidermal growth factor receptor (EGFr) or the closely related p185(erbB2) can promote cell proliferation and survival and thereby contribute to tumorigenesis. Specific antibodies and low molecular-weight tyrosine kinase inhibitors of both proteins are in clinical trials for cancer treatment. CP-654577 is a potent inhibitor selective for p185(erbB2), relative to EGFr tyrosine kinase, and selectively reduces erbB2 autophosphorylation in intact cells. Treatment of SKBr3 human breast cancer cells with CP-654577 reduces the levels of the activated form of mitogen-activated protein kinase, increases the levels of cyclin-dependent kinase inhibitor p27(kip1) and reduces expression of cyclins D and E. These biochemical changes result in a reduced level of phosphorylated retinoblastoma protein and an inhibition of cell-cycle progression at G(1). Apoptosis is triggered in both SKBr3 and another high erbB2-expressing cell line, BT474, by exposure to 1 micro M CP-654577, but this effect is not observed in MCF7 cells that express low erbB2. Levels of activated Akt, an important positive regulator of cell survival, are reduced within 2 h of exposure to 250 nM CP-654577, and this may contribute to the increased apoptosis. These biochemical effects are distinct from those produced by Tarceva, a selective EGFr inhibitor. The antitumor activity of CP-654577 was investigated in athymic mice bearing s.c. tumors from Fischer rat embryo fibroblasts transfected with erbB2. CP-654577 produced a dose-dependent reduction of p185(erbB2) autophosphorylation and inhibited the growth of these tumors. CP-654577 warrants further evaluation in tumors with high expression of p185(erbB2) and may differ from selective EGFr inhibitors or nonselective dual EGFr/erbB2 inhibitors in efficacy and therapeutic index.

1. UK-78,282, a novel piperidine blocker of the T lymphocyte voltage-gated K+ channel, Kv1.3, was discovered by screening a large compound file using a high-throughput 86Rb efflux assay. This compound blocks Kv1.3 with a IC50 of approximately 200 nM and 1:1 stoichiometry. A closely related compound, CP-190,325, containing a benzyl moiety in place of the benzhydryl in UK-78,282, is significantly less potent. 2 Three lines of evidence indicate that UK-78,282 inhibits Kv1.3 in a use-dependent manner by preferentially blocking and binding to the C-type inactivated state of the channel. Increasing the fraction of inactivated channels by holding the membrane potential at - 50 mV enhances the channel's sensitivity to UK-78,282. Decreasing the number of inactivated channels by exposure to approximately 160 mM external K+ decreases the sensitivity to UK-78,282. Mutations that alter the rate of C-type inactivation also change the channel's sensitivity to UK-78,282 and there is a direct correlation between tau(h) and IC50 values. 3. Competition experiments suggest that UK-78,282 binds to residues at the inner surface of the channel overlapping the site of action of verapamil. Internal tetraethylammonium and external charybdotoxin do not compete UK-78,282's action on the channel. 4. UK-78,282 displays marked selectivity for Kv1.3 over several other closely related K+ channels, the only exception being the rapidly inactivating voltage-gated K+ channel, Kv1.4. 5. UK-78,282 effectively suppresses human T-lymphocyte activation.