Crystal structure of the motor domain of centromere-associated protein E in complex with a non-hydrolysable ATP analogue.

Centromere-associated protein E (CENP-E) is a kinesin motor protein essential for mitosis and a new target for anticancer agents with less side effects. To rationally design anticancer drug candidates based on structure, it is important to determine the three-dimensional structure of the CENP-E motor domain bound to its inhibitor. Here, we report the first crystal structure of the CENP-E motor domain in complex with a non-hydrolysable ATP analogue, adenylyl-imidodiphosphate (AMPPNP). Furthermore, the structure is compared with the ADP-bound form of the CENP-E motor domain as well as the AMPPNP-bound forms of other kinesins. This study indicates that helix α4 of CENP-E participates in the slow binding of CENP-E to microtubules. These results will contribute to the development of anticancer drugs targeting CENP-E.

Various effects of two types of kinesin-5 inhibitors on mitosis and cell proliferation.

Kinesin-5 has received considerable attention as a new target for mitosis. Various small-molecule compounds targeting kinesin-5 have been developed in the last few decades. However, the differences in the cellular effects of kinesin-5 inhibitors remain poorly understood. Here, we used two different kinesin-5 inhibitors, biphenyl-type PVZB1194 and S-trityl-L-cysteine-type PVEI0021, to examine their effects on molecular events involving kinesin-5. Our biochemical study of kinesin-5 protein-protein interactions showed that PVZB1194-treated kinesin-5 interacted with TPX2 microtubule nucleation factor, Aurora-A kinase, receptor for hyaluronan-mediated motility, and γ-tubulin, as did untreated mitotic kinesin-5. However, PVEI0021 prevented kinesin-5 from binding to these proteins. In mitotic HeLa cells recovered from nocodazole inhibition, kinesin-5 colocalized with these binding proteins, along with microtubules nucleated near kinetochores. By acting on kinesin-5 interactions with chromatin-associated microtubules, PVZB1194, rather than PVEI0021, not only affected the formation of dispersed microtubule clusters but also enhanced the stability of microtubules. In addition, screening for mitotic inhibitors working synergistically with the kinesin-5 inhibitors revealed that paclitaxel synergistically inhibited HeLa cell proliferation only with PVZB1194. In contrast, the Aurora-A inhibitor MLN8237 exerted a synergistic anti-cell proliferation effect when combined with either inhibitor. Together, these results have provided a better understanding of the molecular action of kinesin-5 inhibitors and indicate their usefulness as molecular tools for the study of mitosis and the development of anticancer agents.

Structure and comparison of the motor domain of centromere-associated protein E.

Centromere-associated protein E (CENP-E) plays an essential role in mitosis and is a target candidate for anticancer drugs. However, it is difficult to design small-molecule inhibitors of CENP-E kinesin motor ATPase activity owing to a lack of structural information on the CENP-E motor domain in complex with its inhibitors. Here, the CENP-E motor domain was crystallized in the presence of an ATP-competitive inhibitor and the crystal structure was determined at 1.9 Šresolution. In the determined structure, ADP was observed instead of the inhibitor in the nucleotide-binding site, even though no ADP was added during protein preparation. Structural comparison with the structures of previously reported CENP-E and those of other kinesins indicates that the determined structure is nearly identical except for several loop regions. However, the retention of ADP in the nucleotide-binding site of the structure strengthens the biochemical view that the release of ADP is a rate-limiting step in the ATPase cycle of CENP-E. These results will contribute to the development of anticancer drugs targeting CENP-E and to understanding the function of kinesin motor domains.

Design, synthesis, and evaluation of a novel prodrug, a S-trityl-l-cysteine derivative targeting kinesin spindle protein.

Kinesin spindle protein (KSP) is expressed only in cells undergoing cell division, and hence represents an attractive target for the treatment of cancer. Several KSP inhibitors have been developed and undergone clinical trial, but their clinical use is limited by their toxicity to rapidly proliferating non-cancerous cells. To create new KSP inhibitors that are highly selective for cancer cells, we optimized the amino acid moiety of S-trityl-l-cysteine (STLC) derivative 1 using in silico modeling. Molecular docking and molecular dynamics simulation were performed to investigate the binding mode of 1 with KSP. Consistent with the structure activity relationship studies, we found that a cysteine amino moiety plays an important role in stabilizing the interaction. Based on these findings and the structure of GSH, a substrate of γ-glutamyltransferase (GGT), we designed and synthesized the prodrug N-γ-glutamylated STLC derivative 9, which could be hydrolyzed by GGT to produce 1. The KSP ATPase inhibitory activity of 9 was lower than that of 1, and LC-MS analysis indicated that 9 was converted to 1 only in the presence of GGT in vitro. In addition, the cytotoxic activity of 9 was significantly attenuated in GGT-knockdown A549 cells. Since GGT is overexpressed on the cell membrane of various cancer cells, these results suggest that compound 9 could be a promising prodrug that selectively inhibits the proliferation of GGT-expressing cancer cells.

Identification of benzo[d]pyrrolo[2,1-b]thiazole derivatives as CENP-E inhibitors.

Kinesin centromere-associated protein E (CENP-E) has emerged as a potential target for the development of anticancer drugs due to its involvement in the mitotic progression of the cell cycle. Although several CENP-E inhibitors have been reported, more knowledge of chemical structures and inhibitory mechanisms is necessary for developing CENP-E inhibitors. Here, we describe the identification of new CENP-E inhibitors. Screening of a small-molecule chemical library identified benzo[d]pyrrolo[2,1-b]thiazole derivatives, including 1, as compounds with inhibitory activity against the microtubule-stimulated ATPase of the CENP-E motor domain. Among the mitotic kinesins examined, 1 selectively inhibited the kinesin ATPase activity of CENP-E. In a steady-state ATPase assay, 1 exhibited ATP-competitive behavior, which was different from the CENP-E inhibitor GSK923295. Compound 1 inhibited the proliferation of tumor-derived HeLa and HCT116 cells more efficiently than that of non-cancerous WI-38 cells. The inhibition of cell proliferation was attributed to the ability of 1 to induce apoptotic cell death. The compound showed antimitotic activity, which caused cell cycle arrest at mitosis via interference with proper chromosome alignment. We identified 1 and its derivatives as the lead compounds that target CENP-E, thus providing a new opportunity for the development of anticancer agents targeting kinesins.

Selective Inhibition of Spindle Microtubules by a Tubulin-Binding Quinazoline Derivative.

In the research field of tubulin-binding agents for the development of anticancer agents, hidden targets are emerging as a problem in understanding the exact mechanisms of actions. The quinazoline derivative 1-(4-methoxyphenyl)-1-(quinazolin-4-yl)ethan-1-ol (PVHD121) has anti-cell proliferative activity and inhibits tubulin polymerization by binding to the colchicine site of tubulin. However, the molecular mechanism of action of PVHD121 in cells remains unclear. Here, we demonstrate that PVHD121 delays mitotic entry and efficiently causes mitotic arrest with spindle checkpoint activation, leading to subsequent cell death. The dominant phenotype induced by PVHD121 was aberrant spindles with robust microtubules and unseparated centrosomes. The microtubules were radially distributed, and their ends appeared to adhere to kinetochores, and not to centrosomes. Extensive inhibition by high concentrations of PVHD121 eliminated all microtubules from cells. PVHD277 [1-(4-methoxyphenyl)-1-(2-morpholinoquinazolin-4-yl)ethan-1-ol], a PVHD121 derivative with fluorescence, tended to localize close to the centrosomes when cells prepared to enter mitosis. Our results show that PVHD121 is an antimitotic agent that selectively disturbs microtubule formation at centrosomes during mitosis. This antimitotic activity can be attributed to the targeting of centrosome maturation in addition to the interference with microtubule dynamics. Due to its unique bioactivity, PVHD121 is a potential tool for studying the molecular biology of mitosis and a potential lead compound for the development of anticancer agents. SIGNIFICANCE STATEMENT: Many tubulin-binding agents have been developed as potential anticancer agents. The aim of this study was to understand the subcellular molecular actions of a quinazoline derivative tubulin-binding agent, 1-(4-methoxyphenyl)-1-(quinazolin-4-yl)ethan-1-ol (PVHD121). As expected from its binding activity to tubulin, PVHD121 caused aberrant spindles and inhibited mitotic progression. However, in addition to tubulin, PVHD121 also targeted an unexpected biomolecule involved in centrosome maturation. Due to targeting the biomolecule just before entering mitosis, PVHD121 preferentially inhibited centrosome-derived microtubules rather than chromosome-derived microtubules during spindle formation. This study not only revealed the molecular action of PVHD121 in cells but also emphasized the importance of considering possible tubulin-independent effects of tubulin-binding agents via hidden targeted biomolecules for future use.

SLCO1B1 Polymorphism Is a Drug Response Predictive Marker for Advanced Pancreatic Cancer Patients Treated With Gemcitabine, S-1, or Gemcitabine Plus S-1.

OBJECTIVES: The aim of this study was to evaluate the effects of single-nucleotide polymorphisms (SNPs) on advanced pancreatic cancer risk and overall survival (OS) in a candidate-gene approach. METHODS: Overall, 5438 SNPs in 219 candidate genes encoding several drug-metabolizing enzymes or transporters were analyzed. In the screening study, 3 SNPs were found associated with OS (P ≤ 0.0005). We validated these SNPs as part of the randomized phase 3 study (GEST study). The associations between OS and SNPs were investigated using log-rank test and Cox proportional hazards model. RESULTS: From the GEST study, the SNP rs4149086 in the 3' UTR of the solute carrier organic anion transporter family member 1B1 (SLCO1B1) gene showed significant interaction with treatment (P = 0.02). In the gemcitabine group, the SNP was associated with short OS (hazard ratio [HR], 3.75; 95% confidence interval [CI], 1.30-10.8; P = 0.008) even after multiple-comparisons adjustment. In contrast, the SNP was not associated with OS in S-1 (HR, 0.77; 95% CI, 0.33-1.81; P = 0.55) or gemcitabine plus S-1 groups (HR, 1.18; 95% CI, 0.46-3.00; P = 0.72). CONCLUSIONS: Patients with advanced pancreatic cancer with the rs4149086 AG or GG genotype may obtain good clinical results when treated with S-1-containing regimens.

Structural and Thermodynamic Basis of the Enhanced Interaction between Kinesin Spindle Protein Eg5 and STLC-type Inhibitors.

For a better understanding of protein-inhibitor interactions, we report structural, thermodynamic, and biological analyses of the interactions between S-trityl-l-cysteine (STLC) derivatives and the motor domain of kinesin spindle protein Eg5. Binding of STLC-type inhibitors to Eg5 was enthalpically driven and entropically unfavorable. The introduction of a para-methoxy substituent in one phenyl ring of STLC enhances its inhibitory activity resulting from a larger enthalpy gain possibly due to the increased shape complementarity. The substituent fits to a recess in the binding pocket. To avoid steric hindrance, the substituted STLC is nudged toward the side opposite to the recess, which enhances the interaction of Eg5 with the remaining part of the inhibitor. Further introduction of an ethylene linkage between two phenyl rings enhances Eg5 inhibitory activity by reducing the loss of entropy in forming the complex. This study provides valuable examples of enhancing protein-inhibitor interactions without forming additional hydrogen bonds.

Investigating toxicity specific to adjuvanted vaccines.

In an attempt to understand the unique toxicity of adjuvanted vaccines, we studied how toxicity develops over time following vaccine administration. In addition to on- and off-target toxicity typically observed with general pharmaceuticals, we observed toxicity associated with both the generation and the broad action of effectors (antibodies and/or cytotoxic T lymphocytes, CTLs). The impact on effector generation appears to be related to local tolerance specific to the adjuvant. The vaccine immune response by effectors serves to demonstrate species relevance as outlined in the recent WHO guideline on the nonclinical evaluation of adjuvanted vaccines. When regarded as pharmaceuticals that function at sites of local administration, adjuvants have inherent on- and off-target toxicity. On-target toxicity of the adjuvant is typically associated with effector generation, and could vary depending on animal species. Therefore, the use of species with sensitivity to adjuvants described in the WHO guidelines is required to evaluate the toxicity of the vaccine associated with effector generation. Changes in safety pharmacology endpoints would be considered off-target and further studies are conducted only if changes in these endpoints are observed in nonclinical or clinical studies. Thus our decision tree does not recommend the routine conduct of stand-alone safety pharmacology studies.

Multicomponent Assembly of the Kinesin Spindle Protein Inhibitor CPUYJ039 and Analogues as Antimitotic Agents.

The potent kinesin spindle protein inhibitor CPUYJ039 and a set of analogues were prepared by a target-oriented approach based on a Ugi reaction that uses 2-nitrophenyl isocyanides as key building blocks. The herein documented strategy provides straightforward and atom economical access to potent benzimidazole-based antimitotic agents by exploring the versatility and exploratory power of the Ugi reaction. The results of docking studies and biological activity evaluations of the benzimidazole compounds are also reported.

Functional 1,3a,6a-triazapentalene scaffold: Design of fluorescent probes for kinesin spindle protein (KSP).

1,3a,6a-Triazapentalene is a compact fluorescent chromophore. In this study, triazapentalene was used to modify a series of biphenyl-type inhibitors of kinesin spindle protein (KSP) to develop fluorescent probes for the intracellular visualization of this protein. Microscopic studies demonstrated that these novel triazapentalene-labeled compounds exhibited inhibitory activity towards KSP in cultured cells and provided important information concerning the intracellular distribution.

Construction of possible integrated predictive index based on EGFR and ANXA3 polymorphisms for chemotherapy response in fluoropyrimidine-treated Japanese gastric cancer patients using a bioinformatic method.

BACKGROUND: Variability in drug response between individual patients is a serious concern in medicine. To identify single-nucleotide polymorphisms (SNPs) related to drug response variability, many genome-wide association studies have been conducted. METHODS: We previously applied a knowledge-based bioinformatic approach to a pharmacogenomics study in which 119 fluoropyrimidine-treated gastric cancer patients were genotyped at 109,365 SNPs using the Illumina Human-1 BeadChip. We identified the SNP rs2293347 in the human epidermal growth factor receptor (EGFR) gene as a novel genetic factor related to chemotherapeutic response. In the present study, we reanalyzed these hypothesis-free genomic data using extended knowledge. RESULTS: We identified rs2867461 in annexin A3 (ANXA3) gene as another candidate. Using logistic regression, we confirmed that the performance of the rs2867461 + rs2293347 model was superior to those of the single factor models. Furthermore, we propose a novel integrated predictive index (iEA) based on these two polymorphisms in EGFR and ANXA3. The p value for iEA was 1.47 × 10(-8) by Fisher's exact test. Recent studies showed that the mutations in EGFR is associated with high expression of dihydropyrimidine dehydrogenase, which is an inactivating and rate-limiting enzyme for fluoropyrimidine, and suggested that the combination of chemotherapy with fluoropyrimidine and EGFR-targeting agents is effective against EGFR-overexpressing gastric tumors, while ANXA3 overexpression confers resistance to tyrosine kinase inhibitors targeting the EGFR pathway. CONCLUSIONS: These results suggest that the iEA index or a combination of polymorphisms in EGFR and ANXA3 may serve as predictive factors of drug response, and therefore could be useful for optimal selection of chemotherapy regimens.

Structure-Guided Design of Novel l-Cysteine Derivatives as Potent KSP Inhibitors.

Kinesin spindle protein (KSP), known as Hs Eg5, a member of the kinesin-5 family, plays an important role in the formation and maintenance of the bipolar spindle. We previously reported S-trityl-l-cysteine derivatives as selective KSP inhibitors. Here, we report further optimizations using docking modeling in the L5 allosteric binding site, which led to the discovery of several high affinity derivatives with two fused phenyl rings in the trityl group giving low nanomolar range KSP ATPase inhibition. The representative derivatives potently inhibited cell growth of HCT116 cells in correlation with KSP inhibitory activities and significantly suppressed tumor growth in the xenograft model in vivo.

Structural basis of new allosteric inhibition in Kinesin spindle protein Eg5.

Kinesin spindle protein Eg5 is a target for anticancer therapies, and small molecule inhibitors of its ATPase activity have been developed. We herein report for the first time the crystal structure of and biochemical studies on the Eg5 motor domain in complex with a new type of allosteric inhibitor. The biphenyl-type inhibitor PVZB1194 binds to the α4/α6 allosteric pocket 15 Å from the ATP-binding pocket, which differs from conventional allosteric inhibitors that bind to the allosteric L5/α2/α3 pocket of Eg5. Binding of the inhibitor is involved in the neck-linker conformation and also causes conformational changes around the ATP-binding pocket through Tyr104 to affect the interaction of ATP with the pocket. This structure provides useful information for the development of novel types of allosteric drugs as well as a novel insight into the molecular mechanism responsible for regulating the motor activity of kinesins.

Application of a combination of a knowledge-based algorithm and 2-stage screening to hypothesis-free genomic data on irinotecan-treated patients for identification of a candidate single nucleotide polymorphism related to an adverse effect.

Interindividual variation in a drug response among patients is known to cause serious problems in medicine. Genomic information has been proposed as the basis for "personalized" health care. The genome-wide association study (GWAS) is a powerful technique for examining single nucleotide polymorphisms (SNPs) and their relationship with drug response variation; however, when using only GWAS, it often happens that no useful SNPs are identified due to multiple testing problems. Therefore, in a previous study, we proposed a combined method consisting of a knowledge-based algorithm, 2 stages of screening, and a permutation test for identifying SNPs. In the present study, we applied this method to a pharmacogenomics study where 109,365 SNPs were genotyped using Illumina Human-1 BeadChip in 168 cancer patients treated with irinotecan chemotherapy. We identified the SNP rs9351963 in potassium voltage-gated channel subfamily KQT member 5 (KCNQ5) as a candidate factor related to incidence of irinotecan-induced diarrhea. The p value for rs9351963 was 3.31×10-5 in Fisher's exact test and 0.0289 in the permutation test (when multiple testing problems were corrected). Additionally, rs9351963 was clearly superior to the clinical parameters and the model involving rs9351963 showed sensitivity of 77.8% and specificity of 57.6% in the evaluation by means of logistic regression. Recent studies showed that KCNQ4 and KCNQ5 genes encode members of the M channel expressed in gastrointestinal smooth muscle and suggested that these genes are associated with irritable bowel syndrome and similar peristalsis diseases. These results suggest that rs9351963 in KCNQ5 is a possible predictive factor of incidence of diarrhea in cancer patients treated with irinotecan chemotherapy and for selecting chemotherapy regimens, such as irinotecan alone or a combination of irinotecan with a KCNQ5 opener. Nonetheless, clinical importance of rs9351963 should be further elucidated.

Considerations for non-clinical safety studies of therapeutic peptide vaccines.

Guidelines for non-clinical studies of prophylactic vaccines against infectious diseases have been published widely, but similar guidelines for therapeutic vaccines, and especially therapeutic peptide vaccines, have yet to be established. The approach to non-clinical safety studies required for therapeutic vaccines differs from that for prophylactic vaccines due to differences in the risk-benefit balance and the mechanisms of action. We propose the following guidelines for non-clinical safety studies for therapeutic peptide vaccines. (i) Since the main safety concern is related to the immune response that might occur at normal sites that express a target antigen, identification of these possible target sites using in silico human expression data is important. (ii) Due to the strong dependence on HLA, it is not feasible to replicate immune responses in animals. Thus, the required non-clinical safety studies are characterized as those detecting off-target toxicity rather than on-target toxicity.

Kinesin spindle protein inhibitors with diaryl amine scaffolds: crystal packing analysis for improved aqueous solubility.

Diaryl amine derivatives have been designed and synthesized as novel kinesin spindle protein (KSP) inhibitors based on planar carbazole-type KSP inhibitors with poor aqueous solubility. The new generation of inhibitors was found to show comparable inhibitory activity and high selectivity for KSP, and this was accompanied with improved solubility. Kinetic analysis and molecular modeling studies suggested that these inhibitors work in an ATP-competitive manner via binding to the secondary allosteric site formed by α4 and α6 helices of KSP. Comparative structural investigations on a series of compounds revealed that the higher solubility of diaryl amine-type inhibitors was attributed to fewer van der Waals interactions in the crystal packing and the hydrogen-bond acceptor nitrogen of the aniline moiety for favorable solvation.

Optimization of diaryl amine derivatives as kinesin spindle protein inhibitors.

Structure-activity relationship studies of diaryl amine-type KSP inhibitors were carried out. Diaryl amine derivatives with a pyridine ring or urea group were less active when compared with the parent carboline and carbazole derivatives. Optimization studies of a lactam-fused diphenylamine-type KSP inhibitor revealed that the aniline NH group and 3-CF3 phenyl group were indispensable for potent KSP inhibition. Modification with a seven-membered lactam-fused phenyl group and a 4-(trifluoromethyl)pyridin-2-yl group improved aqueous solubility while maintaining potent KSP inhibitory activity. From these studies, we identified novel diaryl amine-type KSP inhibitors with a favorable balance of potency and solubility.

Distal promoter regions are responsible for differential regulation of human orosomucoid-1 and -2 gene expression and acute phase responses.

Human orosomucoid (ORM) is a major acute-phase plasma protein, encoded by 2 highly homologous genes, ORM1 and ORM2. Human ORM induction is assumed to be regulated by each proximal promoter region, where putative glucocorticoid responsive elements and CCAAT/enhancer binding protein (C/EBP)ß binding sites are located. However, the details of the differential regulation of these genes remain unknown. To explore this, we assessed the role of the distal promoter region of each ORM in HeLa cells. Luciferase-reporter activities of full constructs, containing approximately 1.1 kbp (FULL), and those of deletion constructs, containing up to 188 bp region (DEL) upstream of the transcription start sites of ORM1 and ORM2 were compared under both basal and inducer-treated conditions. For ORM1 and ORM2 DEL constructs, significantly increased activities after dexamethasone (DEX) treatments (alone and combined with interleukin (IL)-1ß) were observed. Significantly higher FULL construct activities than DEL construct activities were observed for ORM1 after IL-1ß treatment, while those for ORM2 were significantly lower at basal level and after DEX treatments. Upon C/EBPß overexpression, FULL construct activities were significantly higher than those of DEL constructs at basal level and after IL-1ß treatment for ORM1, and at basal level and after inducer-treatments for ORM2. Higher transcription-induction activity in the distal promoter region was evident for ORM1 in the absence of C/EBPß overexpression, and for ORM2 under C/EBPß overexpression conditions. These findings suggest that the ORM distal promoter region differentially regulates expression of ORM genes at basal level and in acute phase responses.

Silk gland factor-2, involved in fibroin gene transcription, consists of LIM homeodomain, LIM-interacting, and single-stranded DNA-binding proteins.

SGF-2 binds to promoter elements governing posterior silk gland-specific expression of the fibroin gene in Bombyx mori. We purified SGF-2 and showed that SGF-2 contains at least four gene products: the silkworm orthologues of LIM homeodomain protein Awh, LIM domain-binding protein (Ldb), a sequence-specific single-stranded DNA-binding protein (Lcaf), and the silk protein P25/fibrohexamerin (fhx). Using co-expression of these factors in Sf9 cells, Awh, Ldb, and Lcaf proteins were co-purified as a ternary complex that bound to the enhancer sequence in vitro. Lcaf interacts with Ldb as well as Awh through the conserved regions to mediate transcriptional activation in yeast. Misexpression of Awh in transgenic silkworms induces ectopic expression of the fibroin gene in the middle silk glands, where Ldb and Lcaf are expressed. Taken together, this study demonstrates that SGF-2 is a multisubunit activator complex containing Awh. Moreover, our results suggest that the Ldb·Lcaf protein complex serves as a scaffold to facilitate communication between transcriptional control elements.