Objectives

Tyrosine kinase inhibitor (TKI)-treated acute myeloid leukemia (AML) patients commonly show rapid and significant peripheral blood blast cell reduction, however a marginal decrease in bone marrow blasts. This suggests a protective environment and highlights the demand for a better understanding of stromal:leukemia cell communication. As a strategy to improve clinical efficacy, we searched for novel agents capable of potentiating the stroma-diminished effects of TKI treatment of mutant FLT3-expressing cells.

Methods

We designed a combinatorial high throughput drug screen using well-characterized kinase inhibitor-focused libraries to identify novel kinase inhibitors capable of overriding stromal-mediated resistance to TKIs, such as PKC412 and AC220. Standard liquid culture proliferation assays, cell cycle and apoptosis analysis, and immunoblotting were carried out with cell lines or primary AML to validate putative candidates from the screen and characterize the mechanism(s) underlying observed synergy.

Results and Conclusions

Our study led to the observation of synergy between selective Akt inhibitors and FLT3 inhibitors against mutant FLT3-positive AML in either the absence or presence of stroma. Our findings are consistent with evidence that Akt activation is characteristic of mutant FLT3-transformed cells, as well as observed residual Akt activity following FLT3 inhibitor treatment. In conclusion, our study highlights the potential importance of Akt as a signaling factor in leukemia survival, and supports the use of the co-culture chemical screen to identify agents able to potentiate TKI anti-leukemia activity in a cytoprotective microenvironment.

The mitogen activated kinases JNK1/2/3 are key enzymes in signaling modules that transduce and integrate extracellular stimuli into coordinated cellular response. Here we report the discovery of the first irreversible inhibitors of JNK1/2/3. We describe two JNK3 co-crystal structures at 2.60 and 2.97 Å resolutions that show the compounds form covalent bonds with a conserved cysteine residue. JNK-IN-8 is a selective JNK inhibitor that inhibits phosphorylation of c-Jun, a direct substrate of JNK kinase, in cells exposed to sub-micromolar drug in a manner that depends on covalent modification of the conserved cysteine residue. Extensive biochemical, cellular and pathway-based profiling establish the selectivity of JNK-IN-8 for JNK and suggest that the compound will be broadly useful as a pharmacological probe of JNK-dependent signal transduction. Potential lead compounds have also been identified for kinases including IRAK1, PIK3C3, PIP4K2C, and PIP5K3.

Foods high in resistant starch (RS) are beneficial to prevent various diseases including diabetes, colon cancers, diarrhea and chronic renal or hepatic diseases. Elevated RS in rice is important for public health since rice is a staple food for half of the world population. A japonica mutant ‘Jiangtangdao 1’ (RS = 11.67%) was crossed with an indica cultivar ‘Miyang 23’ (RS = 0.41%). The mutant sbe3-rs that explained 60.4% of RS variation was mapped between RM6611 and RM13366 on chromosome 2 (LOD = 36) using 178 F2 plants genotyped with 106 genome-wide polymorphic SSR markers. Using 656 plants from four F3∶4 families, sbe3-rs was fine mapped to a 573.3 Kb region between InDel 2 and InDel 6 using one STS, five SSRs and seven InDel markers. SBE3 which codes for starch branching enzyme was identified as a candidate gene within the putative region. Nine pairs of primers covering 22 exons were designed to sequence genomic DNA of the wild type for SBE3 and the mutant for sbe3-rs comparatively. Sequence analysis identified a missense mutation site where Leu-599 of the wild was changed to Pro-599 of the mutant in the SBE3 coding region. Because the point mutation resulted in the loss of a restriction enzyme site, sbe3-rs was not digested by a CAPS marker for SpeI site while SBE3 was. Co-segregation of the digestion pattern with RS content among 178 F2 plants further supported sbe3-rs responsible for RS in rice. As a result, the CAPS marker could be used in marker-assisted breeding to develop rice cultivars with elevated RS which is otherwise difficult to accurately assess in crops. Transgenic technology should be employed for a definitive conclusion of the sbe3-rs.

Summary

The mechanical properties of organic and biomolecular thin films on surfaces play an important role in a broad range of applications. Although force-modulation microscopy (FMM) is used to map the apparent elastic properties of such films with high lateral resolution in air, it has rarely been applied in aqueous media. In this letter we describe the use of FMM to map the apparent elastic properties of self-assembled monolayers and end-tethered protein thin films in aqueous media. Furthermore, we describe a simple analysis of the contact mechanics that enables the selection of FMM imaging parameters and thus yields a reliable interpretation of the FMM image contrast.

Summary

Protein kinases are intensely studied mediators of cellular signaling, yet important questions remain regarding their regulation and in vivo properties. Here we use a probe-based chemoprotemics platform to profile several well studied kinase inhibitors against more than 200 kinases in native cell proteomes and reveal new biological targets for some of these inhibitors. Several striking differences were identified between native and recombinant kinase inhibitory profiles, in particular, for the Raf kinases. The native kinase binding profiles presented here closely mirror the cellular activity of these inhibitors, even when the inhibition profiles differ dramatically from recombinant assay results. Additionally, Raf activation events could be detected upon live cell treatment with inhibitors. These studies highlight the complexities of protein kinase behavior in the cellular context and demonstrate that profiling with only recombinant/purified enzymes can be misleading.

While genomically targeted therapies have improved outcomes for patients with lung adenocarcinoma, little is known about the genomic alterations which drive squamous cell lung cancer. Sanger sequencing of the tyrosine kinome identified mutations in the DDR2 kinase gene in 3.8% of squamous cell lung cancers and cell lines. Squamous lung cancer cell lines harboring DDR2 mutations were selectively killed by knock-down of DDR2 by RNAi or by treatment with the multi-targeted kinase inhibitor dasatinib. Tumors established from a DDR2 mutant cell line were sensitive to dasatinib in xenograft models. Expression of mutated DDR2 led to cellular transformation which was blocked by dasatinib. A squamous cell lung cancer patient with a response to dasatinib and erlotinib treatment harbored a DDR2 kinase domain mutation. These data suggest that gain-of-function mutations in DDR2 are important oncogenic events and are amenable to therapy with dasatinib. As dasatinib is already approved for use, these findings could be rapidly translated into clinical trials.

A series of alkyne containing type II inhibitors with potent inhibitory activity of T315I Bcr-Abl has been identified. The most active compound 4 exhibits an EC50 of less than 1 nM against wild-type Bcr-Abl and an EC50 of 10 nM against T315I mutant but is broadly active against a number of other kinases.

Summary

The fibroblast growth factor receptor tyrosine kinases (FGFR1, 2, 3, and 4) represent promising therapeutic targets in a number of cancers. We have developed the first potent and selective irreversible inhibitor of FGFR1, 2, 3, and 4 which we named FIIN-1 that forms a covalent bond with cysteine 486 located in the P-loop of the FGFR1 ATP-binding site. We demonstrate that the inhibitor potently inhibits Tel-FGFR1 transformed Ba/F3 cells (EC50 = 14 nM) as well as numerous FGFR-dependent cancer cell lines. A biotin-derivatized version of the inhibitor, FIIN-1-biotin, was shown to covalently label FGFR1 at Cys486. FIIN-1 is a useful probe of FGFR-dependent cellular phenomena and may provide a starting point of the development of therapeutically relevant irreversible inhibitors of wild-type and drug-resistant forms of FGFR kinases.

SUMMARY

In an effort to find new pharmacological modalities to overcome resistance to ATP-site inhibitors of Bcr-Abl, we recently reported the discovery of GNF-2, a selective allosteric Bcr-Abl inhibitor. Here, using solution NMR, X-ray crystallography, mutagenesis and hydrogen exchange mass spectrometry we demonstrate that GNF-2 binds to the myristate binding site of Abl, leading to changes in the structural dynamics of the ATP-binding site. GNF-5, an analog of GNF-2 having improved pharmacokinetic properties, when utilized in combination with the ATP-competitive inhibitors imatinib or nilotinib, suppressed the emergence of resistance mutations in vitro, displayed additive inhibitory activity in biochemical and cellular assays against T315I Bcr-Abl and displayed in vivo efficacy against the recalcitrant T315I Bcr-Abl mutant in a murine bone-marrow transplantation model. These results demonstrate that therapeutically relevant inhibition of Bcr-Abl activity can be achieved using inhibitors that bind to the myristate binding site and that combining allosteric and ATP-competitive inhibitors can overcome resistance to either agent alone.

Abl kinase inhibitors targeting the ATP binding pocket are currently employed as potent anti-leukemogenic agents but drug resistance has become a significant clinical limitation. Recently, a compound that binds to the myristate pocket of Abl (GNF-5) was shown to act cooperatively with nilotinib, an ATP-competitive inhibitor to target the recalcitrant “T315I” gatekeeper mutant of Bcr-Abl. To uncover an explanation for how drug binding at a distance from the kinase active site could lead to inhibition and how inhibitors could combine their effects, hydrogen exchange mass spectrometry (HX MS) was employed to monitor conformational effects in the presence of both dasatinib, a clinically approved ATP-site inhibitor, and GNF-5. While dasatinib binding to wild type Abl clearly influenced Abl conformation, no binding was detected between dasatinib and T315I. GNF-5, however, elicited the same conformational changes in both wild type and T315I, including changes to dynamics within the ATP site located approximately 25 Å from the site of GNF-5 interaction. Simultaneous binding of dasatinib and GNF-5 to T315I caused conformational and/or dynamics changes in Abl such that effects of dasatinib on T315I were the same as when it bound to wild type Abl. These results provide strong biophysical evidence that allosteric interactions play a role in Abl kinase downregulation and that targeting sites outside the ATP binding site can provide an important pharmacological tool to overcome mutations that cause resistance to ATP-competitive inhibitors.

A polymer brush consisting of 70% poly(N-isopropylacrylamide) (PNIPAAM) and 30% polymethacrylic acid (PMAA) was synthesized from gold substrates with a grafting-from AIBN type free-radical initiator. Fractionation of two peptides, Bradykinin and Buccalin, was accomplished in less than 120 seconds by placing a 30 pM (pH∼6.2) droplet onto the polymer brush substrate. The eluant containing the anionic Buccalin is pipetted away for MALDI analysis while the cationic Bradykinin adsorbed to the swollen anionic brush and was subsequently released by adding a droplet of formic acid to the substrate. This caused the brush to collapse and release the Bradykinin, much like squeezing a sponge; these nanosponge substrates exhibited very high loading capacity (>2.0 mg/ml) compared to plasma-polymer-modified MALDI substrates. Ellipsometric measurements showed that complementary peptides adsorb rapidly while those of the same charge do not and MALDI-MS analysis of the two fractions showed separation of both peptides. The adsorption of Bradykinin was monitored over time and 85% of the peptide had been adsorbed to the nanosponge in 1 minute from a 0.5 mg/ml aqueous solution.

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.

In the title mol­ecule, C27H18ClNO3, the anthracene mean plane forms dihedral angles of 67.43 (2) and 15.75 (3)° with the isoxazole and benzene rings, respectively. In the crystal structure, C—H⋯π inter­actions link mol­ecules into centrosymmetric dimers, which are further linked by weak inter­molecular C—H⋯N hydrogen bonds into ribbons propagating in the [110] direction.

Neuroblastoma, an embryonal tumor of the peripheral sympathetic nervous system, accounts for approximately 15% of all deaths due to childhood cancer1. High-risk neuroblastomas, prevalent in the majority of patients, are rapidly progressive; even with intensive myeloablative chemotherapy, relapse is common and almost uniformly fatal2,3. Here we report the detection of previously unknown mutations in the ALK gene, which encodes a receptor tyrosine kinase, in 8% of primary neuroblastomas. Five non-synonymous sequence variations were identified in the kinase domain of ALK, of which three were somatic and two were germline. The most frequent mutation, F1174L, was also identified in three different neuroblastoma cell lines. ALK cDNAs encoding the F1174L and R1275Q variants, but not the wild-type ALK cDNA, transformed IL-3-dependent murine hematopoietic Ba/F3 cells to cytokine-independent growth. Ba/F3 cells expressing these mutations were sensitive to a small-molecule inhibitor of ALK, TAE6844. Furthermore, two human neuroblastoma cell lines harboring the F1174L mutation were sensitive to the inhibitor. Cytotoxicity was associated with increased levels of apoptosis as measured by TUNEL-labeling. shRNA-mediated knockdown of ALK expression in neuroblastoma cell lines with the F1174L mutation also resulted in apoptosis and impaired cell proliferation. Thus, activating alleles of the ALK receptor tyrosine kinase are present in primary neuroblastoma tumors and in established neuroblastoma cell lines, and confer sensitivity to ALK inhibition with small molecules, providing a molecular rationale for targeted therapy of this disease.

In the title compound, [Mn(C5H6N3O)2(C3H7NO)]ClO4, the MnIII atom lies on the inversion centre of the centrosymmetric complex cation and has a distorted octa­hedral coordination geometry, formed by two N atoms and two O atoms from two 1-methyl­imidazole-2-carbaldehyde oximate ligands and two O atoms from two dimethyl­formamide ligands. Perchlorate acts as a counterion to balance the charge. The crystal structure of the title compound is stabilized by C—H⋯O hydrogen-bonding inter­actions.

Inhibition of Bcr-Abl kinase activity by imatinib for the treatment of chronic myeloid leukemia (CML) currently serves as the paradigm for targeting dominant oncogenes with small molecules. We recently reported the discovery of GNF-2 (1) and GNF-5 (2) as selective non-ATP competitive inhibitors of cellular Bcr-Abl kinase activity that target the myristate binding site. Here, we used cell-based structure−activity relationships to guide the optimization and diversification of ligands that are capable of binding to the myristate binding site and rationalize the findings based upon an Abl−compound 1 cocrystal. We elucidate the structure−activity relationships required to obtain potent antiproliferative activity against Bcr-Abl transformed cells and report the discovery of new compounds (5g, 5h, 6a, 14d, and 21j-I) that display improved potency or pharmacological properties. This work demonstrates that a variety of structures can effectively target the Bcr-Abl myristate binding site and provides new leads for developing drugs that can target this binding site.

A series of novel 3,5-diamino-1,2,4-triazole benzyl ureas was identified as having potent anaplastic lymphoma kinase (ALK) inhibition exemplified by 15a, 20a, and 23a, which exhibited antiproliferative IC50 values of 70, 40, and 20 nM in Tel-ALK transformed Ba/F3 cells, respectively. Moreover, 15a and 23a potently inhibited the growth and survival of NPM-ALK positive anaplastic large cell lymphoma cell (SU-DHL-1) and neuroblastoma cell lines (KELLY, SH-SY5Y) containing the F1174L ALK mutation. These compounds provide novel leads for the development of small-molecule ALK inhibitors for cancer therapy.