A Compound I Mimic Reveals the Transient Active Species of a Cytochrome P450 Enzyme: Insight into the Stereoselectivity of P450-Catalysed Oxidations.

Catching the structure of cytochrome P450 enzymes in flagrante is crucial for the development of P450 biocatalysts, as most structures collected are found trapped in a precatalytic conformation. At the heart of P450 catalysis lies Cpd I, a short-lived, highly reactive intermediate, whose recalcitrant nature has thwarted most attempts at capturing catalytically relevant poses of P450s. We report the crystal structure of P450BM3 mimicking the state in the precise moment preceding epoxidation, which is in perfect agreement with the experimentally observed stereoselectivity. This structure was attained by incorporation of the stable Cpd I mimic oxomolybdenum mesoporphyrin IX into P450BM3 in the presence of styrene. The orientation of styrene to the Mo-oxo species in the crystal structures sheds light onto the dynamics involved in the rotation of styrene to present its vinyl group to Cpd I. This method serves as a powerful tool for predicting and modelling the stereoselectivity of P450 reactions.

Size-Based Differentiation of Cancer and Normal Cells by a Particle Size Analyzer Assisted by a Cell-Recognition PC Software.

Detection of anomalous cells such as cancer cells from normal blood cells has the potential to contribute greatly to cancer diagnosis and therapy. Conventional methods for the detection of cancer cells are usually tedious and cumbersome. Herein, we report on the use of a particle size analyzer for the convenient size-based differentiation of cancer cells from normal cells. Measurements made using a particle size analyzer revealed that size parameters for cancer cells are significantly greater (e.g., inner diameter and width) than the corresponding values for normal cells (white blood cells (WBC), lymphocytes and splenocytes), with no significant difference in shape parameters (e.g., circularity and convexity). The inner diameter of many cancer cell lines is greater than 10 µm, in contrast to normal cells. For the detection of WBC having similar size to that of cancer cells, we developed a PC software "Cancer Cell Finder" that differentiates them from cancer cells based on brightness stationary points on a cell surface. Furthermore, the aforementioned method was validated for cancer cell/clusters detection in spiked mouse blood samples (a B16 melanoma mouse xenograft model) and circulating tumor cell cluster-like particles in the cat and dog (diagnosed with cancer) blood samples. These results provide insights into the possible applicability of the use of a particle size analyzer in conjunction with PC software for the convenient detection of cancer cells in experimental and clinical samples for theranostics.

Site-Specific Dual Functionalization of Cysteine Residue in Peptides and Proteins with 2-Azidoacrylates.

Herein, we report use of 2-azidoacrylates to perform site-specific dual functionalization of the cysteine residue of peptides and bovine serum albumin (BSA), a native protein containing one free cysteine residue. The sulfhydryl group of the cysteine residue could be conjugated with 2-azidoacrylates bearing various functionalities, such as fluorescent dyes under physiological aqueous buffer conditions, to afford peptide and protein conjugates anchoring an azide moiety. Successive azide-alkyne cycloaddition enables installation of the second functionality, thus affording dual-functionalized peptide- and protein-based materials.

Investigation of Thermally Induced Cellular Ablation and Heat Response Triggered by Planar MoS2-Based Nanocomposite.

In comparison to conventional tumor treatment methods, photothermal therapy (PTT) is one of the innovative therapeutic strategies that employs light to produce localized heat for targeted ablation of cancer cells. Among the various kinds of heat generation nanomaterials, transition metal dichalcogenide nanosheets, especially molybdenum disulfide (MoS2), have recently been investigated as one of the promising PTT candidates because of their strong absorbance in the near-infrared (NIR) tissue transparency window and excellent photothermal conversion capability. In line with the great potential of MoS2-based nanomaterials in biomedical applications, their intrinsic therapeutic performance and corresponding cellular response are required to be continually investigated. In order to further improve MoS2-based PTT efficacy and dissect the molecular mechanism during heat stimuli, in this study, we successfully designed a novel and effective PTT platform by integration of MoS2 nanosheets with peptide-based inhibition molecules to block the function of heat shock proteins (Hsp90), one type of chaperone proteins that play protective roles in living systems against cellular photothermal response. Such a combined nanosystem could effectively induce cell ablation and viability assays indicated approximately 5-fold higher PTT treatment efficacy (8.8% viability) than that of MoS2 itself (48% viability) upon 808 nm light irradiation. Moreover, different from the case based on MoS2 alone that could cause tumor ablation through the process of necrosis, the detailed mechanism analysis revealed that the inhibition of Hsp90 could significantly increase the photothermally mediated apoptosis, hence resulting in remarkable enhancement of photothermal treatment. Such promising studies provide the great opportunity to better understand the cellular basis of light-triggered thermal response. Moreover, they can also facilitate the rational design of new generations of PTT platforms toward future theranostics.

Development of a novel sulfonate ester-based prodrug strategy.

A self-immolative γ-aminopropylsulfonate linker was investigated for use in the development of prodrugs that are reactive to various chemical or biological stimuli. To demonstrate their utility, a leucine-conjugated prodrug of 5-chloroquinolin-8-ol (5-Cl-8-HQ), which is a potent inhibitor against aminopeptidase from Aeromonas proteolytica (AAP), was synthesized. The sulfonate prodrug was considerably stable under physiological conditions, with only enzyme-mediated hydrolysis of leucine triggering the subsequent intramolecular cyclization to simultaneously release 5-Cl-8-HQ and form γ-sultam. It was also confirmed that this γ-aminopropylsulfonate linker was applicable for prodrugs of not only 8-HQ derivatives but also other drugs bearing a phenolic hydroxy group.

Enrichment of circulating tumor cells in tumor-bearing mouse blood by a deterministic lateral displacement microfluidic device.

Concentration of real tumor cells leaking into blood from cancer was attempted by a deterministic lateral displacement (DLD) microfluidic device. Spiked cultured cell line tumor cells are often used to verify performance of the circulating tumor cells (CTCs) separation methods. Cultured tumor cells are obviously larger than most of hematocytes and considered not to be appropriate as CTC mimics, while there is uncertainty in identifying real CTCs from clinical samples and there is no practical way to examine CTCs leakage into benign cells during the sorting. In this work, blood samples were prepared from tumor-bearing mice whose tumors were induced by implanting cells with GFP expression to living mice. Therefore, CTCs were identified by their fluorescence emission. We succeeded in the enrichment of tumor cells to 0.05% from the blood, in which CTCs were negligibly detected among three million blood cells, and little loss of CTCs was observed.

AS-2, a novel inhibitor of p53-dependent apoptosis, prevents apoptotic mitochondrial dysfunction in a transcription-independent manner and protects mice from a lethal dose of ionizing radiation.

In a previous study, we reported that some tetradentate zinc(II) chelators inhibit p53 through the denaturation of its zinc-requiring structure but a chelator, Bispicen, a potent inhibitor of in vitro apoptosis, failed to show any efficient radioprotective effect against irradiated mice because the toxicity of the chelator to mice. The unsuitability of using tetradentate chelators as radioprotectors prompted us to undertake a more extensive search for p53-inhibiting agents that are weaker zinc(II) chelators and therefore less toxic. Here, we show that an 8-hydroxyquinoline (8HQ) derivative, AS-2, suppresses p53-dependent apoptosis through a transcription-independent mechanism. A mechanistic study using cells with different p53 characteristics revealed that the suppressive effect of AS-2 on apoptosis is specifically mediated through p53. In addition, AS-2 was less effective in preventing p53-mediated transcription-dependent events than pifithrin-µ (PFTµ), an inhibitor of transcription-independent apoptosis by p53. Fluorescence visualization of the extranuclear distribution of AS-2 also supports that it is ineffective on the transcription-dependent pathway. Further investigations revealed that AS-2 suppressed mitochondrial apoptotic events, such as the mitochondrial release of intermembrane proteins and the loss of mitochondrial membrane potential, although AS-2 resulted in an increase in the mitochondrial translocation of p53 as opposed to the decrease of cytosolic p53, and did not affect the apoptotic interaction of p53 with Bcl-2. AS-2 also protected mice that had been exposed to a lethal dose of ionizing radiation. Our findings indicate that some types of bidentate 8HQ chelators could serve as radioprotectors with no substantial toxicity in vivo.

Design and synthesis of 8-hydroxyquinoline-based radioprotective agents.

In radiation therapy, adverse side effects are often induced due to the excessive cell death that occurs in radiosensitive normal cells. The radiation-induced cell death of normal cells is caused, at least in part, by apoptosis, which undergoes via activation of p53 and increase in the p53 protein, a zinc-containing transcriptional factor, in response to cellular damage. Therefore, radioprotective drugs that can protect normal cells from radiation and thus suppress adverse side effects would be highly desirable. We report herein on the radioprotective activity of 8-hydroxyquinoline (8HQ) derivatives that were initially designed so as to interact with the Zn(2+) in p53. Indeed, the 5,7-bis(methylaminosulfonyl)-8HQ and 8-methoxyquinoline derivatives considerably protected MOLT-4 cells against γ-ray radiation (10 Gy), accompanied by a low cytotoxicity. However, mechanistic studies revealed that the interaction of these drugs with p53 is weak and the mechanism for inhibiting apoptosis appears to be different from that of previously reported radioprotectors such as bispicen, which inhibits apoptosis via the denaturation of p53 as well as by blocking both transcription-dependent and -independent apoptotic pathways.

Design, synthesis and photochemical reactivation of caged prodrugs of 8-hydroxyquinoline-based enzyme inhibitors.

8-Hydroxyquinoline (HQ)-based compounds have recently been proposed as potential candidates for drugs for treating human immunodeficiency virus (HIV), cancer, neurodegenerative diseases (Alzheimer's and Parkinson's disease), and parasitic and bacterial infections. However, HQ itself and its derivatives might be toxic due to their intrinsic affinity for metal cations in living systems. One possible strategy for suppressing the toxicity and side effects of drugs with metal chelation properties, such as HQ, would be masking the critically important moieties with protecting groups that can be subsequently removed under specific conditions. In our previous work, we reported that HQ analogs are potent and selective inhibitors (Ki values=0.16-29 µM) of aminopeptidase from Aeromonas proteolytica (AAP) (EC 3.4.11.10), a dinuclear Zn(2+) peptidase. Based on this background information, HQ sulfonates were synthesized as prodrugs of HQ-based AAP-inhibitors that can be reactivated by photochemical cleavage of the S-O bond in the sulfonate groups. The findings indicate that HQ sulfonates containing methanesulfonyl and 2-aminoethanesulfonyl groups are essentially stable under physiological conditions and undergo photolysis to regenerate the corresponding HQ compounds that function as AAP inhibitors. This methodology could be applied to the design of similar types of Zn(2+) hydrolase inhibitors and prodrugs.

Heme-substituted protein assembly bridged by synthetic porphyrin: achieving controlled configuration while maintaining rotational freedom.

The use of biological host-guest interactions, specifically the binding of hemoprotein to heme, has attracted significant research interest in the design of artificial protein assemblies. However, because of the inherent flexibility of the propionic acid group of heme, it is difficult to control the positioning and orientation of the protein unit and to construct well-ordered structures. Herein, we report a heme-substituted protein dimer composed of the native hemoprotein HasA, which accommodates a tetraphenylporphyrin bearing an additional metal coordination site. The specific binding of the tetraphenylporphyrin with an additional metal coordination site that protrudes in a fixed direction confines the configuration of the dimer structure to a defined bent form. The small-angle X-ray scattering profile shows the dimer structure with a bent form and suggests dynamic rotational behavior while keeping its bent-core structure, resembling a bevel gear. This unique dimer structure demonstrates that the design of heme-substituted protein assemblies can be expanded to protein assemblies while maintaining the rotational freedom of the individual protein units.

Investigating the applicability of the CYP102A1-decoy-molecule system to other members of the CYP102A subfamily.

Cytochrome P450 enzymes (CYPs) have attracted much promise as biocatalysts in a push for cleaner and more environmentally friendly catalytic systems. However, changing the substrate specificity of CYPs, such as CYP102A1, can be a challenging task, requiring laborious mutagenesis. An alternative approach is the use of decoy molecules that "trick" the enzyme into becoming active by impersonating the native substrate. Whilst the decoy molecule system has been extensively developed for CYP102A1, its general applicability for other CYP102-family enzymes has yet to be shown. Herein, we demonstrate that decoy molecules can "trick" CYP102A5 and A7 into becoming active and hydroxylating non-native substrates. Furthermore, significant differences in decoy molecule selectivity as well as decoy molecule binding were observed. The X-ray crystal structure of the CYP102A5 haem domain was solved at 2.8 Å, delivering insight into a potential substate-binding site that differs significantly from CYP102A1.

Selective capture and collection of live target cells using a photoreactive silicon wafer device modified with antibodies via a photocleavable linker.

A device for the capture and recollection of live target cells is described. The platform was a silicon (Si) wafer modified with an anti-HEL antibody (anti-HEL-IgG, HEL = hen egg lysozyme) through a photocleavable 3-amino-3-(2-nitrophenyl)propionic acid (ANP) linker. The modification processes of the Si wafer surface were monitored by Fourier transform infrared spectroscopy-attenuated total reflection (FTIR-ATR) and fast-scanning atomic force microscopy (FS-AFM). The attachment of IgG and its release reaction on the Si surface via the photochemical cleavage of the ANP linker were observed directly by FS-AFM. The results of an enzyme-linked immunosorbent assay (ELISA) indicated that the photorelease of the complex of anti-HEL-IgG with the secondary antibody-alkaline phosphatase hybrid (secondary IgG-AP) from the Si surface occurs with minimum damage. Furthermore, it was possible to collect SP2/O cells selectively that express HEL on their cell membranes (SP2/O-HEL) on the Si wafer device. Photochemical cleavage of the ANP linker facilitated the effective release of living SP2/O cells whose viability was verified by staining experiments using tripan blue. Moreover, it was possible to reculture the recovered cells. This methodology represents an effective strategy for isolating intact target cells in the biological and medicinal sciences and related fields.

Photochemical cleavage reaction of 8-quinolinyl sulfonates that are halogenated and nitrated at the 7-position.

Photochemical bond-cleavage reactions are potentially useful in chemistry, bioorganic chemistry and medicinal chemistry. We previously reported on a photochemical cleavage reaction of 8-quinolinyl sulfonate (8-QS) derivatives in aqueous solution at neutral pH, which we proposed to proceed via an excited triplet state. In this report, we report on the synthesis of some new photocleavable 8-QS derivatives, in which halogen atoms or a nitro group was introduced at the 7-position, in an attempt to improve photoreactive properties and to produce a red-shift in the irradiation wavelength. The introduction of bromine and iodine resulted in an acceleration in the photoreaction by about 1.5 times, possibly due to a heavy atom effect. It was also found that 7-nitro-8-QS absorbs at >360 nm, and, as a result, the S-O bond of this compound can be cleaved by photoirradiation with a fluorescent lamp in aqueous solution and on silicon surface.

Expanding the applicability of cytochrome P450s and other haemoproteins.

Cytochrome P450BM3 has long been regarded as a promising candidate for use as a biocatalyst, owing to its excellent efficiency for the hydroxylation of unactivated C-H bonds. However, because of its high substrate specificity, its possible applications have been severely limited. Consequently, various approaches have been proposed to overcome the enzyme's natural limitations, thereby expanding its substrate scope to encompass non-native substrates, evoking chemoselectivity, regioselectivity and stereoselectivity and enabling previously inaccessible chemical conversions. Herein, these approaches will be classified into three categories: (1) mutagenesis including directed evolution, (2) haem substitution with artificial cofactors and (3) use of substrate mimics, 'decoy molecules'. Herein, we highlight the representative work that has been conducted in above three categories for discussion of the future outlook of P450BM3 in green chemistry.

Alignment of gold clusters on DNA via a DNA-recognizing zinc finger-metallothionein fusion protein.

The complementary recognition of base pairs (bp) is the major strategy in the "DNA lithography" of gold (Au)clusters or nanoparticles, where single-stranded DNAs sulfurized at their termini are generally used to bind Au clusters or nanoparticles. In this report, we discuss a new material that can be used to locate Au clusters on the desired positions of DNA. For this purpose, we combined a two-domain zinc finger (ZF) and the analogue of R domain of rat's liver metallothionein (MT) to utilize the DNA-recognizing ability of ZF motifs and the heavy metal binding ability of MTs, and prepared an artificial fusion protein, ZFZF-MTalpha (1). Titration experiments monitored by absorption and circular dichroism spectroscopies, as well as an electrophoretic mobility shift assay and quantification using 5,5'-dithiobis(2-nitrobenzoic acid), clarified that (1) the ZF domain traps two divalent metal ions to fold in a ZF structure with M(Cys)2(His)2 (M = Co, Zn, and Cd) coordination units, (2) the MT domain traps metal ions to form clusters (Cd2+ particularly forms a Cd4(Cys)9 cluster) without interfering with the folding of the ZF domain, and (3) 1 recognizes the 5'-GGGGGG-3' (G6) bp sequence in the presence of Zn2+ based on the amino acid sequence encoded in the ZF domain. The titration of Au11(PPh3)8Cl3 (Au11) into the solution of 1 in the presence of Zn2+ revealed that the MT domain strongly binds the Au11 cluster with a 1:1 ratio, and a Au11-containing conjugate, ZF(Zn)ZF(Zn)-MTalpha(Au11) was obtained. Transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy, and X-ray photoelectron spectroscopy showed that this conjugate maintains an Au11 core in the form of Au11(PPh3)4(S-Cys)6 without disturbing the folding nature of the ZF domain. ZF(Zn)ZF(Zn)-MTR(Au11) recognizes the bp sequence G6 with K(d1) = 450 nM, while simultaneously forming a dimer on the DNA with K(d2) = 200 nM. TEM experiments showed that the conjugates form parallelograms or triangles (defective parallelograms) on a double crossover (dx) DNA, according to the positions of G6 encoded in the dx DNA.

Minimal motif peptide structure of metzincin clan zinc peptidases in micelles.

It is well known that the functions of metalloproteins generally originate from their metal-binding motifs. However, the intrinsic nature of individual motifs remains unknown, particularly the details about metal-binding effects on the folding of motifs; the converse is also unknown, although there is no doubt that the motif is the core of the reactivity for each metalloprotein. In this study, we focused our attention on the zinc-binding motif of the metzincin clan family, HEXXHXXGXXH; this family contains the general zinc-binding sequence His-Glu-Xaa-Xaa-His (HEXXH) and the extended GXXH region. We adopted the motif sequence of stromelysin-1 and investigated the folding properties of the Trp-labeled peptides WAHEIAHSLGLFHA (STR-W1), AWHEIAHSLGLFHA (STR-W2), AHEIAHSLGWFHA (STR-W11), and AHEIAHSLGLFHWA (STR-W14) in the presence and absence of zinc ions in hydrophobic micellar environments by circular dichroism (CD) measurements. We accessed successful incorporation of these zinc peptides into micelles using quenching of Trp fluorescence. Results of CD studies indicated that two of the Trp-incorporated peptides, STR-W1 and STR-W14, exhibited helical folding in the hydrophobic region of cetyltrimethylammonium chloride micelle. The NMR structural analysis of the apo STR-W14 revealed that the conformation in the C-terminus GXXH region significantly differred between the apo state in the micelle and the reported Zn-bound state of stromelysin-1 in crystal structures. The structural analyses of the qualitative Zn-binding properties of this motif peptide provide an interesting Zn-binding mechanism: the minimum consensus motif in the metzincin clan, a basic zinc-binding motif with an extended GXXH region, has the potential to serve as a preorganized Zn binding scaffold in a hydrophobic environment.

Simple and Convenient Method for the Isolation, Culture, and Re-collection of Cancer Cells from Blood by Using Glass-Bead Filters.

Circulating tumor cells (CTCs) are tumor cells that originate from primary cancer tissues, enter the bloodstream in the body, and metastasize to the other organs. Simple and convenient methods for their detection, capture, and recovery from the blood of cancer patients would be highly desirable. We report here on a simple and convenient methodology to trap, culture, and re-collect cancer cells, the sizes of which are greater than those of normal hematologic cells, by the use of glass-bead filters (GBFs). We prepared GBFs with a diameter of 24 mm and thicknesses of 0.4 mm and 1.2 mm, with well-defined pores, by sintering round-shaped glass beads (diameter: 63-106 µm). A small integrated glass-bead filter (iGBF) with a diameter of ca. 9.6 mm for the use in filtering a small volume of blood was also designed and prepared. Using GBF and iGBF, it was possible to efficiently capture mouse Lewis lung carcinoma cells expressing green fluorescent protein spiked in saline/blood by single and repeated (circulation) filtrations in in vitro experiments with very small amounts of red blood cells being captured. In addition, we successfully captured B16 CTCs from the blood of a B16 melanoma metastasis mouse model by iGBF. Cancer cells/CTCs captured on/in the GBF could be cultured and efficiently recovered from the filters. Filtration by GBF had negligible effect on the adherent and proliferative characteristics of cancer cells. Simple and convenient methods for the capture, culture, and re-collection of CTCs by GBF along with flexibility of GBF, which permits them to be molded into suitable architectures having the desired shape and size, should be useful for early and convenient diagnosis and treatment of cancer and related diseases.

Reconstitution of full-length P450BM3 with an artificial metal complex by utilising the transpeptidase Sortase A.

Haem substitution is an effective approach to tweak the function of haemoproteins. Herein, we report a facile haem substitution method for self-sufficient cytochrome P450BM3 (CYP102A1) from Bacillus megaterium utilising the transpeptidase Sortase A from Staphylococcus aureus. We successfully constructed Mn-substituted BM3 and investigated its catalytic activity.

A Chemical Modulator of p53 Transactivation that Acts as a Radioprotective Agonist.

Inhibiting p53-dependent apoptosis by inhibitors of p53 is an effective strategy for preventing radiation-induced damage in hematopoietic lineages, while p53 and p21 also play radioprotective roles in the gastrointestinal epithelium. We previously identified some zinc(II) chelators, including 8-quinolinol derivatives, that suppress apoptosis in attempts to discover compounds that target the zinc-binding site in p53. We found that 5-chloro-8-quinolinol (5CHQ) has a unique p53-modulating activity that shifts its transactivation from proapoptotic to protective responses, including enhancing p21 induction and suppressing PUMA induction. This p53-modulating activity also influenced p53 and p53-target gene expression in unirradiated cells without inducing DNA damage. The specificity of 5CHQ for p53 and p21 was demonstrated by silencing the expression of each protein. These effects seem to be attributable to the sequence-specific alteration of p53 DNA-binding, as evaluated by chromatin immunoprecipitation and electrophoretic mobility shift assays. In addition, 5-chloro-8-methoxyquinoline itself had no antiapoptotic activity, indicating that the hydroxyl group at the 8-position is required for its antiapoptotic activity. We applied this remarkable agonistic activity to protecting the hematopoietic and gastrointestinal system in mouse irradiation models. The dose reduction factors of 5CHQ in total-body and abdominally irradiated mice were about 1.2 and 1.3, respectively. 5CHQ effectively protected mouse epithelial stem cells from a lethal dose of abdominal irradiation. Furthermore, the specificity of 5CHQ for p53 in reducing the lethality induced by abdominal irradiation was revealed in Trp53-KO mice. These results indicate that the pharmacologic upregulation of radioprotective p53 target genes is an effective strategy for addressing the gastrointestinal syndrome. Mol Cancer Ther; 17(2); 432-42. ©2017 AACRSee all articles in this MCT Focus section, "Developmental Therapeutics in Radiation Oncology."

Glycopeptide antibiotic analogs for selective inactivation and two-photon imaging of vancomycin-resistant strains.

A novel theranostic divalent vancomycin analog using a planar 1,8-diazapyrene moiety as a rigid scaffold exhibits potent and selective antibacterial activity against Gram (+) bacteria including vancomycin-resistant strains, while having minimal influence on Gram (-) bacteria and mammalian cells. Moreover, this theranostic analog can be also applied for selective two-photon fluorescence imaging of Gram (+) bacteria.