Discovery of an Aurora kinase inhibitor through site-specific dynamic combinatorial chemistry

We demonstrate a fragment-based lead discovery method that combines site-directed ligand discovery with dynamic combinatorial chemistry. Our technique targets dynamic combinatorial screening to a specified region of a protein by using reversible disulfide chemistry. We have used this technology to rapidly identify inhibitors of the drug target Aurora A that span the purine-binding site and the adaptive pocket of the kinase. The binding mode of a noncovalent inhibitor has been further characterized through crystallography.     

Dictyostelium Aurora kinase has properties of both Aurora A and Aurora B kinases

Aurora kinases are highly conserved proteins with important roles in mitosis. Metazoans contain two kinases, Aurora A and B, which contribute distinct functions at the spindle poles and the equatorial region respectively. It is not currently known whether the specialized functions of the two kinases arose after their duplication in animal cells or were already present in their ancestral kinase. We show that Dictyostelium discoideum contains a single Aurora kinase, DdAurora, that displays characteristics of both Aurora A and B. Like Aurora A, DdAurora has an extended N-terminal domain with an A-box sequence and localizes at the spindle poles during early mitosis. Like Aurora B, DdAurora binds to its partner DdINCENP and localizes on centromeres at metaphase, the central spindle during anaphase, and the cleavage furrow at the end of cytokinesis. DdAurora also has several unusual properties. DdAurora remains associated with centromeres in anaphase, and this association does not require an interaction with DdINCENP. DdAurora then localizes at the cleavage furrow, but only at the end of cytokinesis. This localization is dependent on DdINCENP and the motor proteins Kif12 and myosin II. Thus, DdAurora may represent the ancestral kinase that gave rise to the different Aurora kinases in animals and also those in other organisms.     

Water-soluble prodrugs of cyclosporine A with tailored conversion rates.

A novel type of water-soluble prodrugs of cyclosporine A (CsA) is described, featuring a modular system of an enzyme-cleavable group, a solubilizing moiety and a chemodegradable spacer attached to the hydroxyl function of (4R)-4-[(E)-2-butenyl]-4-,N-dimethyl-l-threonine (MeBmt)-1 of CsA. The chemical synthesis of these double prodrugs proceeds in high yield and purity and allows for a systematic study of the influence of the structural parameters upon physicochemical and pharmacological properties. The evaluation of the chemical and enzymatic stability results in differential values of the conversion rates (minutes to several hours) in support of an enzyme-triggered release of the parent drug as the rate-limiting step. In vitro studies show that the designed prodrug systems can be regarded as soft prodrugs in being devoid of cyclophiline A (CypA) binding and that complete conversion to the parent drug occurs in whole rat blood, setting the stage for therapeutic use.     

Synthesis and evaluation of N-acyl sulfonamides as potential prodrugs of cyclin-dependent kinase inhibitor JNJ-7706621

A novel prodrug strategy for cyclin-dependent kinase inhibitor JNJ-7706621 has been explored. Through N-acylation of a sulfonamide substituent, tails containing different solubilizing groups (amino, carboxyl, alkoxyl, and hydroxyl) were attached to JNJ-7706621. Most of the prodrugs exhibited good aqueous solubility and the N-acyl groups on the sulfonamide were metabolically cleaved to generate active drug in rat PK study.     

Controlled drug release: new water-soluble prodrugs of an HIV protease inhibitor

We designed and synthesized a series of highly water-soluble prodrugs of an HIV protease inhibitor, KNI-727 (1), containing tandem-linked two auxiliary units, a solubilizing moiety and a self-cleavable spacer. Prodrugs with an ionized amino group at the solubilizing moiety exhibited a remarkable increase of water-solubility (>10(4) fold) compared to the parent drug 1. These prodrugs released I not enzymatically, but chemically via an intramolecular cyclization-elimination reaction through an imide formation in physiological conditions. Diversified rates of parent drug release were observed when the chemical structure of both the solubilizing and the spacer moieties were modified. This new approach for water-soluble prodrugs will enable to control chemically the release of parent drug as well as to maintain high water-solubility.     

有丝分裂激酶Aurora B的研究进展

真核生物细胞通过有丝分裂将遗传物质均等地分配到两个子细胞中,从而维持基因组的稳定性。有丝分裂的每一环节都需要精准而细致的调控,这依赖于一系列调节机制,尤其需要多个相关激酶的共同协调。Aurora B是一个关键的有丝分裂调控激酶,伴随有丝分裂的进行,其先后在染色体臂、内着丝粒、中央纺锤体、中体上动态分布。与其高度时空动态性相一致的是,Aurora B在有丝分裂的多个环节,如姐妹染色体粘连、动粒微管连接、纺锤体检验点和胞质分裂过程中都发挥着一系列重要功能。本文将概述近年来Aurora B激酶功能与调控方面的研究进展。     

SAR and inhibitor complex structure determination of a novel class of potent and specific Aurora kinase inhibitors

A novel series of 5-aminopyrimidinyl quinazolines has been developed from anilino-quinazoline 1, which was identified in a high throughput screen for Aurora A. Introduction of the pyrimidine ring and optimisation of the substituents both on this ring and at the C7 position of the quinazoline led to the discovery of compounds that are highly specific Aurora kinase inhibitors. Co-crystallisation of one of these inhibitors with a fragment of Aurora A shows the importance of the benzamido group in achieving selectivity.     

2-Aminobenzimidazoles as potent Aurora kinase inhibitors

This Letter describes the discovery and key structure–activity relationship (SAR) of a series of 2-aminobenzimidazoles as potent Aurora kinase inhibitors. 2-Aminobenzimidazole serves as a bioisostere of the biaryl urea residue of SNS-314 (1c), which is a potent Aurora kinase inhibitor and entered clinical testing in patients with solid tumors. Compared to SNS-314, this series of compounds offers better aqueous solubility while retaining comparable in vitro potency in biochemical and cell-based assays; in particular, 6m has also demonstrated a comparable mouse iv PK profile to SNS-314.     

Uncovering new substrates for Aurora A kinase

Aurora A is a serine/threonine kinase that is essential for a wide variety of cell-cycle-related events, but only a small number of its substrates are known. We present and validate a strategy by which to identify Aurora A substrates and their phosphorylation sites. We developed a computational approach integrating various types of biological information to generate a list of 90 potential Aurora substrates, with a prediction accuracy of about 80%. We also demonstrated the specific phosphorylation of NUSAP (nucleolar and spindle-associated protein) by Aurora A in vivo. Our results provide a means by which to develop an understanding of Aurora A function and suggest unexpected roles for this kinase.     

The Aurora B kinase inhibitor AZD1152 sensitizes cancer cells to fractionated irradiation and induces mitotic catastrophe

AZD1152, an Aurora kinase inhibitor with selectivity for Aurora B kinase, can enhance the effect of ionizing radiation (IR). The aim of this study was to evaluate and to mechanistically explore scheduling effects of AZD1152 on tumor responses to IR, in three different settings: neoadjuvant (AZD1152 before IR), adjuvant (IR before AZD1152), or concomitant treatments (AZD1152 plus one single IR dose). A more pronounced tumor growth delay was observed in the neoadjuvant and adjuvant schedules as compared to the concomitant schedule. However, AZD1152 enhanced the efficacy of IR when concomitant IR was fractionated over several days. Histopathological examination revealed that AZD1152 + IR induced polyploidy, multinucleation and micronuclei in vivo. Time-lapse videomicroscopy confirmed that cell death induced by AZD1152 + IR was preceded by multinucleation and the formation of micronuclei, which both are hallmarks of mitotic catastrophe. Caspase inhibition or removal of the pro-apoptotic protein Bax did not ameliorate the long-term cell survival of AZD1152-treated cancer cells. In contrast, a chemical inhibitor of CHK1, Chir124, sensitized cancer cells to the lethal effect of AZD1152. Altogether, these data support the contention that AZD1152 mediates radiosensitization in vivo by enhancing mitotic catastrophe, which can be used as a biomarker of treatment efficacy.     

Aurora B kinase inhibition in mitosis: Strategies for optimizing the use of Aurora kinase inhibitors such as AT9283

Aurora kinases play a key role in regulating mitotic division and are attractive oncology targets. AT9283, a multi-targeted kinase inhibitor with potent activity against Aurora A and B kinases, inhibited growth and survival of multiple solid tumor cell lines and was efficacious in mouse xenograft models. AT9283-treatment resulted in endoreduplication and ablation of serine-10 histone H3 phosphorylation in both cells and tumor samples, confirming that in these models it acts as an Aurora B kinase inhibitor. In vitro studies demonstrated that exposure to AT9283 for one complete cell cycle committed an entire population of p53 checkpoint-compromised cells (HCT116) to multinucleation and death whereas treatment of p53 checkpoint-competent cells (HMEC, A549) for a similar length of time led to a reversible arrest of cells with 4N DNA. Further studies in synchronized cell populations suggested that exposure to AT9283 during mitosis was critical for optimal cytotoxicity. We therefore investigated ways in which these properties might be exploited to optimize the efficacy and therapeutic index of Aurora kinase inhibitors for p53 checkpoint compromised tumors in vivo. Combining Aurora B kinase inhibition with paclitaxel, which arrests cells in mitosis, in a xenograft model resulted in promising efficacy without additional toxicity. These findings have implications for optimizing the efficacy of Aurora kinase inhibitors in clinical practice.     

The induction of polyploidy or apoptosis by the Aurora A kinase inhibitor MK8745 is p53-dependent

Aurora kinases are mitotic serine/threonine protein kinases and are attractive novel targets for anticancer therapy. Many small-molecule inhibitors of Aurora kinases are currently undergoing clinical trials. Aurora A kinase is essential for successful mitotic transition. MK8745 is a novel and selective small-molecule inhibitor of Aurora A kinase. MK8745 induced apoptotic cell death in a p53-dependent manner when tested in vitro in cell lines of multiple lineages. Cells expressing wild-type p53 showed a short delay in mitosis followed by cytokinesis, resulting in 2N cells along with apoptosis. However, cells lacking or with mutant p53 resulted in a prolonged arrest in mitosis followed by endoreduplication and polyploidy. Cytokinesis was completely inhibited in p53-deficient cells, as observed by the absence of 2N cell population. The induction of apoptosis in p53-proficient cells was associated with activation of caspase 3 and release of cytochrome c but was independent of p21. Exposure of p53 wild-type cells to MK8745 resulted in the induction of p53 phosphorylation (ser15) and an increase in p53 protein expression. p53-dependent apoptosis by MK8745 was further confirmed in HCT 116 p53^−/− cells transfected with wild-type p53. Transient knockdown of Aurora A by specific siRNA recapitulated these p53-dependent effects, with greater percent induction of apoptosis in p53 wild-type cells. In conclusion, our studies show p53 as a determining factor for induction of apoptosis vs. polyploidy upon inhibition of Aurora A.Key words: Aurora A kinase, polyploidy, apoptosis, p53, cell cycle     

The induction of polyploidy or apoptosis by the Aurora A kinase inhibitor MK8745 is p53-dependent

Aurora kinases are mitotic serine/threonine protein kinases and are attractive novel targets for anticancer therapy. Many small-molecule inhibitors of Aurora kinases are currently undergoing clinical trials. Aurora A kinase is essential for successful mitotic transition. MK8745 is a novel and selective small-molecule inhibitor of Aurora A kinase. MK8745 induced apoptotic cell death in a p53-dependent manner when tested in vitro in cell lines of multiple lineages. Cells expressing wild-type p53 showed a short delay in mitosis followed by cytokinesis, resulting in 2N cells along with apoptosis. However, cells lacking or with mutant p53 resulted in a prolonged arrest in mitosis followed by endoreduplication and polyploidy. Cytokinesis was completely inhibited in p53-deficient cells, as observed by the absence of 2N cell population. The induction of apoptosis in p53-proficient cells was associated with activation of caspase 3 and release of cytochrome c but was independent of p21. Exposure of p53 wild-type cells to MK8745 resulted in the induction of p53 phosphorylation (ser15) and an increase in p53 protein expression. p53-dependent apoptosis by MK8745 was further confirmed in HCT 116 p53-/- cells transfected with wild-type p53. Transient knockdown of Aurora A by specific siRNA recapitulated these p53- dependent effects, with greater percent induction of apoptosis in p53 wild-type cells. In conclusion, our studies show p53 as a determining factor for induction of apoptosis vs. polyploidy upon inhibition of Aurora A.     

A functional cooperativity between Aurora A kinase and LIM kinase1

Aurora kinase A (Aur-A), a mitotic kinase, regulates initiation of mitosis through centrosome separation and proper assembly of bipolar spindles. LIM kinase 1 (LIMK1), a modulator of actin and microtubule dynamics, is involved in the mitotic process through inactivating phosphorylation of cofilin. Phosphorylated LIMK1 is recruited to the centrosomes during early prophase, where it colocalizes with γ-tubulin. Here, we report a novel functional cooperativity between Aur-A and LIMK1 through mutual phosphorylation. LIMK1 is recruited to the centrosomes during early prophase and then to the spindle poles, where it colocalizes with Aur-A. Aur-A physically associates with LIMK1 and activates it through phosphorylation, which is important for its centrosomal and spindle pole localization. Aur-A also acts as a substrate of LIMK1, and the function of LIMK1 is important for its specific localization and regulation of spindle morphology. Taken together, the novel molecular interaction between these two kinases and their regulatory roles on one another’s function may provide new insight on the role of Aur-A in manipulation of actin and microtubular structures during spindle formation.     

Tetraploidization increases sensitivity to Aurora B kinase inhibition

Aurora kinases are overexpressed in many cancers and are targets for anticancer drugs. The yeast homolog of Aurora B kinase, IPL1, was found to be a ploidy-specific lethality gene. Given that polyploidization is a common feature of many cancers, we hypothesized polyploidization also sensitizes mammalian cells to inhibition of Aurora kinases. Using two models of apparent diploid vs. tetraploid cell lines (one based on the hepatocellular carcinoma cell line Hep3B and another on untransformed mouse fibroblasts), we found that tetraploid cells were more sensitive to Aurora B inhibition than their diploid counterparts. Apoptosis could be induced in tetraploid cells by two different Aurora B inhibitors. Furthermore, tetraploid cells were sensitive to Aurora B inhibition but were not affected by Aurora A inhibition. Interestingly, the underlying mechanism was due to mitotic slippage and the subsequent excessive genome reduplication. In support of this, abolition of cytokinesis with dihydrocytochalasin B resulted in similar effects on tetraploid cells as Aurora B inhibition. These results indicate that inhibition of Aurora B or cytokinesis can promote apoptosis effectively in polyploid cancer cells.     

Characterization of a splicing variant of plant Aurora kinase

Aurora kinases play a key role in chromosome segregation and cytokinesis. In plants, three Aurora kinases (AtAUR1-AtAUR3) have been identified in Arabidopsis thaliana. Here, we report an AtAUR2 splicing variant (AtAUR2S), which lacks the fourth exon encoding a part of the kinase domain of AtAUR2. AtAUR2S was shown to have lost its kinase activity to phosphorylate histone H3 at Ser10; however, it maintained its ability to bind to histone H3. The localization pattern of AtAUR2S was the same as that of AtAUR2. The findings suggest that AtAUR2S affects cell division by competing with AtAUR2.