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Pancreatic ductal adenocarcinoma (PDAC) is characterized by hypoxic niches that lead to treatment resistance. Therefore, studies of tumor oxygenation and metabolic profiling should contribute to improved treatment strategies. Here, we define two imaging biomarkers that predict differences in tumor response to therapy: (i) partial oxygen pressure (pO2), measured by EPR imaging; and (ii) [1-13C] pyruvate metabolism rate, measured by hyperpolarized 13C MRI. Three human PDAC xenografts with varying treatment sensitivity (Hs766t, MiaPaCa2, and Su.86.86) were grown in mice. The median pO2 of the mature Hs766t, MiaPaCa2, and Su.86.86 tumors was 9.1 ± 1.7, 11.1 ± 2.2, and 17.6 ± 2.6 mm Hg, and the rate of pyruvate-to-lactate conversion was 2.72 ± 0.48, 2.28 ± 0.26, and 1.98 ± 0.51 per minute, respectively (n = 6, each). These results are in agreement with steady-state data of matabolites quantified by mass spectroscopy and histologic analysis, indicating glycolytic and hypoxia profile in Hs766t, MiaPaca2, and Su.86.86 tumors. Fractionated radiotherapy (5 Gy × 5) resulted in a tumor growth delay of 16.7 ± 1.6 and 18.0 ± 2.7 days in MiaPaca2 and Su.86.86 tumors, respectively, compared with 6.3 ± 2.7 days in hypoxic Hs766t tumors. Treatment with gemcitabine, a first-line chemotherapeutic agent, or the hypoxia-activated prodrug TH-302 was more effective against Hs766t tumors (20.0 ± 3.5 and 25.0 ± 7.7 days increase in survival time, respectively) than MiaPaCa2 (2.7 ± 0.4 and 6.7 ± 0.7 days) and Su.86.86 (4.7 ± 0.6 and 0.7 ± 0.6 days) tumors. Collectively, these results demonstrate the ability of molecular imaging biomarkers to predict the response of PDAC to treatment with radiotherapy and TH-302.Significance: pO2 imaging data and clinically available metabolic imaging data provide useful insight into predicting the treatment efficacy of chemotherapy, radiation, and a hypoxia-activated prodrug as monotherapies and combination therapies in PDAC tumor xenograft models. Cancer Res; 78(14); 3783–92. ©2018 AACR.

Plots of tumor growth delay vs HF10; pO2; lac/pyr

Supplemental materials and methods

Median pO2 and HF10 of individual PDAC tumors in EPR oxygen imaging study.

Supplemental Figures

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Purpose: To evaluate the potential of hyperpolarized [1-13C]-pyruvate magnetic resonance spectroscopic imaging (MRSI) of prostate cancer as a predictive biomarker for targeting the Warburg effect.Experimental Design: Two human prostate cancer cell lines (DU145 and PC3) were grown as xenografts. The conversion of pyruvate to lactate in xenografts was measured with hyperpolarized [1-13C]-pyruvate MRSI after systemic delivery of [1-13C] pyruvic acid. Steady-state metabolomic analysis of xenograft tumors was performed with mass spectrometry and steady-state lactate concentrations were measured with proton (1H) MRS. Perfusion and oxygenation of xenografts were measured with electron paramagnetic resonance (EPR) imaging with OX063. Tumor growth was assessed after lactate dehydrogenase (LDH) inhibition with FX-11 (42 μg/mouse/day for 5 days × 2 weekly cycles). Lactate production, pyruvate uptake, extracellular acidification rates, and oxygen consumption of the prostate cancer cell lines were analyzed in vitro. LDH activity was assessed in tumor homogenates.Results: DU145 tumors demonstrated an enhanced conversion of pyruvate to lactate with hyperpolarized [1-13C]-pyruvate MRSI compared with PC3 and a corresponding greater sensitivity to LDH inhibition. No difference was observed between PC3 and DU145 xenografts in steady-state measures of pyruvate fermentation, oxygenation, or perfusion. The two cell lines exhibited similar sensitivity to FX-11 in vitro. LDH activity correlated to FX-11 sensitivity.Conclusions: Hyperpolarized [1-13C]-pyruvate MRSI of prostate cancer predicts efficacy of targeting the Warburg effect. Clin Cancer Res; 24(13); 3137–48. ©2018 AACR.

Elucidation of biological phenomena requires imaging of microenvironments in vivo. Although the seamless visualization of in vivo hypoxia from the level of whole-body to single-cell has a great potential to discover unknown phenomena in biological and medical fields, no methodology for achieving it has been established thus far. Here, we for the first time report the whole-body and whole-organ imaging of hypoxia, an important microenvironment, at single-cell resolution using activatable covalent fluorescent probes compatible with tissue clearing. We initially focused on overcoming the incompatibility of fluorescent dyes and refractive index matching solutions (RIMSs), which has greatly hindered the development of fluorescent molecular probes in the field of tissue clearing. The fluorescent dyes compatible with RIMS were then incorporated into the development of activatable covalent fluorescent probes for hypoxia. We combined the probes with tissue clearing, achieving comprehensive single-cell-resolution imaging of hypoxia in a whole mouse body and whole organs.

Reoxygenation has a significant impact on the tumor response to radiotherapy. With developments in radiotherapy technology, the relevance of the reoxygenation phenomenon in treatment efficacy has been a topic of interest. Evaluating the reoxygenation in the tumor microenvironment throughout the course of radiation therapy is important in developing effective treatment strategies. In the current study, we used electron paramagnetic resonance imaging (EPRI) to directly map and quantify the partial oxygen pressure (pO

Our understanding of the mechanism of action of bioactive small molecules contributes to the research and development of new medical drugs, as well as elucidating the pathological mechanisms underlying various diseases. Researchers in this field are committed to a very ambitious goal: the discovery of novel therapeutic compounds along with their molecular targets. To achieve this goal, new methodological developments are indispensable.This review gives an update on the advancements of phage display (PD) technology in the past decade (2011-2020) for determining the targets of the small molecule therapeutics. In particular, other than providing a brief overview of this field of research, we focus on reporting the research trends and the results solely obtained using this strategy.Despite the development of bioinformatics tools and artificial intelligence (AI)-mediated methods, affinity-guided information obtained experimentally are still indispensable to identify drug-protein interactions. By taking advantage of small-molecule-oriented PD methods and their improvements, the extension of the druggable proteome will be further expanded, providing new opportunities to generate small-molecule therapeutics.

Dynamic nuclear polarization (DNP) is a cutting-edge technique that markedly enhances the detection sensitivity of molecules using nuclear magnetic resonance (NMR)/magnetic resonance imaging (MRI). This methodology enables real-time imaging of dynamic metabolic status in vivo using MRI. To expand the targetable metabolic reactions, there is a demand for developing exogenous, i.e., artificially designed, DNP-NMR molecular probes; however, complying with the requirements of practical DNP-NMR molecular probes is challenging because of the lack of established design guidelines. Here, we report Ala-[1- 13 C]Gly- d 2 -NMe 2 as a DNP-NMR molecular probe for in vivo detection of aminopeptidase N activity. We developed this probe rationally through precise structural investigation, calculation, biochemical assessment, and advanced molecular design to achieve rapid and detectable responses to enzyme activity in vivo. With the fabricated probe, we successfully detected enzymatic activity in vivo. This report presents a comprehensive approach for the development of artificially derived, practical DNP-NMR molecular probes through structure-guided molecular design.

Dynamic nuclear polarization (DNP) is a cutting-edge technique that dramatically enhances the detection sensitivity of molecules using nuclear magnetic resonance imaging (NMR/MRI). This methodology enables real-time imaging of dynamic metabolic status in vivo using MRI. To expand the targetable metabolic reactions, there is a demand for developing exogenous, i.e., artificially designed, DNP-NMR molecular probes; however, complying with the requirements of practical DNP-NMR molecular probes is challenging due to the lack of established design guidelines. Herein, we report Ala-[1-13C]Gly-d2-NMe2 as a DNP-NMR molecular probe for in vivo detection of aminopeptidase N (APN) activity. We developed this probe rationally through precise structural investigation, calculation, biochemical assessment, and advanced molecular design to achieve rapid and detectable responses to enzyme activity in vivo. With the fabricated probe, we successfully detected enzymatic activity in vivo. This report presents a comprehensive approach for the development of artificially derived, practical DNP-NMR molecular probes through structure-guided molecular design.

Dissolution-dynamic nuclear polarization (d-DNP) is a state-of-the-art technology that can dramatically enhance the detection sensitivity of nuclear magnetic resonance (NMR). DNP NMR has been applied to small molecules with stable isotopes and has been used to obtain metabolic and physiological information in vivo. However, the hyperpolarized state exponentially decays back to the thermal equilibrium state, depending on the spin-lattice relaxation time (T1). This signal decay has remained a major problem associated with this technology. Therefore, DNP NMR molecular probes useful for in vivo analysis have been limited to naturally occurring small molecules that inherently show long T1. While peptides are promising targets for DNP NMR studies, because of the limitation in T1, DNP NMR molecular probes applicable in vivo have been limited to amino acids or dipeptides. Herein we propose a 13C-labeling strategy to utilize the C-terminal [1-13C]Gly-d2 residue for realizing long T1 in peptides. Structure-based T1 relaxation analysis of amino acids and peptides revealed that (1) T1 does not decrease monotonically with increasing molecular weight and (2) T1 is not significantly affected by a side chain on the neighboring amino acid residue. These findings suggest that the C-terminal [1-13C]Gly-d2 residue affords sufficiently long T1 for biological uses, even in oligopeptides, and allowed us to develop 13C-b- casomorphin-5 (Tyr-Pro-Phe-Pro-[1-13C]Gly-d2, T1 = 24 ± 4 s at 3 T in H2O) and 13C-glutathione (g-Glu-Cys-[1-13C]Gly-d2, T1 = 58 ± 3 s at 3 T in H2O) as DNP NMR probes with long T1. We succeeded in in vivo detection of enzymatic conversions of these two probes. These results demonstrate the utility of our strategy and would contribute to further expansion of the substrate scope for DNP applications.

Serine hydroxymethyltransferase (SHMT) is an enzyme that catalyzes the reaction that converts serine to glycine. It plays an important role in one-carbon metabolism. Recently, SHMT has been shown to be associated with various diseases. Therefore, SHMT has attracted attention as a biomarker and drug target. However, the development of molecular probes responsive to SHMT has not yet been realized. This is because SHMT catalyzes an essential yet simple reaction; thus, the substrates that can be accepted into the active site of SHMT are limited. Here, we focus on the SHMT-catalyzed retro-aldol reaction rather than the canonical serine–glycine conversion and succeed in developing fluorescent and 19F NMR molecular probes. Taking advantage of the facile and direct detection of SHMT, the developed fluorescent probe is used in the high-throughput screening for human SHMT inhibitors, and two hit compounds are obtained., The enzyme serine hydroxymethyltransferase (SHMT) has been implicated in several diseases, however is hard to investigate. Here, the authors used a design strategy based on the retro-aldol-type reaction catalyzed by SHMT to develop SHMT-responsive fluorescence and 19F NMR molecular probes.

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation of the drug-protein interactions is highly desirable for not only understanding the molecular mechanisms underlying therapeutic efficacy but also for assessing drug characteristics, such as adsorption, distribution, metabolism, excretion, and toxicity (ADMET) for clinical use. Here, we present a biosensor-based high throughput strategy for the biopanning of T7 phage-displayed short peptides that can be easily displayed on the phage capsid. Subsequent analysis of the amino acid sequences of peptides containing short segments, as "broken relics", of the drug-binding sites using bioinformatics programs in receptor ligand contact (RELIC) suite, is also shown. By applying this method to two clinically approved drugs, an anti-tumor irinotecan, and an anti-flu oseltamivir, the detailed process for collecting the drug-recognizing peptide sequences and highlighting the drug-binding sites of the target proteins are explained in this paper. The strategy described herein can be applied for any small molecules of interest.

The enhanced permeability and retention (EPR) effect is variable depending on nanoparticle properties and tumor/vessel conditions. Thus, intratumoral evaluations of the vasculature and nanoparticle distribution are important for predicting the therapeutic efficacy and the intractability of tumors. We aimed to develop a tumor vasculature evaluation method and high-resolution nanoparticle delivery imaging using magnetic resonance (MR) micro-imaging technology with a gadolinium (Gd)-dendron assembled liposomal contrast agent. Using the Gd-liposome and a cryogenic receiving coil, we achieved 50-μm isotropic MR angiography with clear visualization of tumor micro-vessel structure. The Gd-liposome-enhanced MR micro-imaging revealed differences in the vascular structures between Colon26- and SU-DHL6-grafted mice models. The vessel volumes and diameters measured for both tumors were significantly correlated with histological observations. The MR micro-imaging methods facilitate the evaluation of intratumoral vascularization patterns, the quantitative assessment of vascular-properties that alter tumor malignancy, particle retentivity, and the effects of treatment.

Dynamic nuclear polarization (DNP) is a technique to polarize the nuclear spin population. As a result of the hyperpolarization, the NMR sensitivity of the nuclei in molecules can be dramatically enhanced. Recent application of the hyperpolarization technique has led to advances in biochemical and molecular studies. A major problem is the short lifetime of the polarized nuclear spin state. Generally, in solution, the polarized nuclear spin state decays to a thermal spin equilibrium, resulting in loss of the enhanced NMR signal. This decay is correlated directly with the spin-lattice relaxation time T1 . Here we report [13 C,D14 ]tert-butylbenzene as a new scaffold structure for designing hyperpolarized 13 C probes. Thanks to the minimized spin-lattice relaxation (T1 ) pathways, its water-soluble derivative showed a remarkably long 13 C T1 value and long retention of the hyperpolarized spin state.

Hyperpolarization is an emerging method that dramatically enhances NMR signal intensity. As a result of their increased sensitivity, hyperpolarized (HP) NMR molecular probes can be used to perform time-resolved spectroscopy and imaging in vitro and in vivo. It is, however, challenging to design such probes de novo. Herein, the [1-13 C]α-amino acid is reported as a scaffold structure to design HP 13 C NMR molecular probes. The [1-13 C]α-amino acid can be converted to various HP 13 C chemical probes that show sufficient chemical shift change by altering the chemical state of the α nitrogen upon interaction with the target. Several previously reported HP probes could be explained by this design principle. To demonstrate the versatility of this approach, two α-amino-acid-based HP 13 C chemical probes, sensitive to pH and Ca2+ ion, were developed and used to detect targets.

In hyperpolarization of (13)C-enriched magnetic resonance chemical probes in the solid-state, a trace amount of gadolinium (Gd) contrast agent can be used to maximize polarization of the (13)C nuclear spins. Here, we report systematic measurement of the spin-lattice relaxation time (T1) and enhancement level of (13)C-enriched chemical probes in the presence of various Gd contrast agents in the liquid-state after dissolution. Using two different (13)C probes having opposite electric charges at neutral pH, we clearly show the T1 of hyperpolarized (13)C was barely affected by the use of a Gd complex that displays repulsive interaction with the (13)C probe in solution, whilst T1 was drastically shortened when there was ionic attraction between probe and complex.

Hypoxic zones in solid tumors contribute to radioresistance, and pharmacologic agents that increase tumor oxygenation prior to radiation, including antiangiogenic drugs, can enhance treatment response to radiotherapy. Although such strategies have been applied, imaging assessments of tumor oxygenation to identify an optimum time window for radiotherapy have not been fully explored. In this study, we investigated the effects of α-sulfoquinovosylacyl-1,3-propanediol (SQAP or CG-0321; a synthetic derivative of an antiangiogenic agent) on the tumor microenvironment in terms of oxygen partial pressure (pO2), oxyhemoglobin saturation (sO2), blood perfusion, and microvessel density using electron paramagnetic resonance imaging, photoacoustic imaging, dynamic contrast–enhanced MRI with Gd-DTPA injection, and T2*-weighted imaging with ultrasmall superparamagnetic iron oxide (USPIO) contrast. SCCVII and A549 tumors were grown by injecting tumor cells into the hind legs of mice. Five days of daily radiation (2 Gy) combined with intravenous injection of SQAP (2 mg/kg) 30 minutes prior to irradiation significantly delayed growth of tumor xenografts. Three days of daily treatment improved tumor oxygenation and decreased tumor microvascular density on T2*-weighted images with USPIO, suggesting vascular normalization. Acute effects of SQAP on tumor oxygenation were examined by pO2, sO2, and Gd-DTPA contrast-enhanced imaging. SQAP treatment improved perfusion and tumor pO2 (ΔpO2: 3.1 ± 1.0 mmHg) and was accompanied by decreased sO2 (20%–30% decrease) in SCCVII implants 20–30 minutes after SQAP administration. These results provide evidence that SQAP transiently enhanced tumor oxygenation by facilitating oxygen dissociation from oxyhemoglobin and improving tumor perfusion. Therefore, SQAP-mediated sensitization to radiation in vivo can be attributed to increased tumor oxygenation. Significance: A multimodal molecular imaging study evaluates pharmacological alteration of the tumor microenvironment to improve radiation response.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by hypoxic niches that lead to treatment resistance. Therefore, studies of tumor oxygenation and metabolic profiling should contribute to improved treatment strategies. Here, we define two imaging biomarkers that predict differences in tumor response to therapy: (i) partial oxygen pressure (pO2), measured by EPR imaging; and (ii) [1-13C] pyruvate metabolism rate, measured by hyperpolarized 13C MRI. Three human PDAC xenografts with varying treatment sensitivity (Hs766t, MiaPaCa2, and Su.86.86) were grown in mice. The median pO2 of the mature Hs766t, MiaPaCa2, and Su.86.86 tumors was 9.1 ± 1.7, 11.1 ± 2.2, and 17.6 ± 2.6 mm Hg, and the rate of pyruvate-to-lactate conversion was 2.72 ± 0.48, 2.28 ± 0.26, and 1.98 ± 0.51 per minute, respectively (n = 6, each). These results are in agreement with steady-state data of matabolites quantified by mass spectroscopy and histologic analysis, indicating glycolytic and hypoxia profile in Hs766t, MiaPaca2, and Su.86.86 tumors. Fractionated radiotherapy (5 Gy × 5) resulted in a tumor growth delay of 16.7 ± 1.6 and 18.0 ± 2.7 days in MiaPaca2 and Su.86.86 tumors, respectively, compared with 6.3 ± 2.7 days in hypoxic Hs766t tumors. Treatment with gemcitabine, a first-line chemotherapeutic agent, or the hypoxia-activated prodrug TH-302 was more effective against Hs766t tumors (20.0 ± 3.5 and 25.0 ± 7.7 days increase in survival time, respectively) than MiaPaCa2 (2.7 ± 0.4 and 6.7 ± 0.7 days) and Su.86.86 (4.7 ± 0.6 and 0.7 ± 0.6 days) tumors. Collectively, these results demonstrate the ability of molecular imaging biomarkers to predict the response of PDAC to treatment with radiotherapy and TH-302. Significance: pO2 imaging data and clinically available metabolic imaging data provide useful insight into predicting the treatment efficacy of chemotherapy, radiation, and a hypoxia-activated prodrug as monotherapies and combination therapies in PDAC tumor xenograft models. Cancer Res; 78(14); 3783–92. ©2018 AACR.

Purpose: To evaluate the potential of hyperpolarized [1-13C]-pyruvate magnetic resonance spectroscopic imaging (MRSI) of prostate cancer as a predictive biomarker for targeting the Warburg effect. Experimental Design: Two human prostate cancer cell lines (DU145 and PC3) were grown as xenografts. The conversion of pyruvate to lactate in xenografts was measured with hyperpolarized [1-13C]-pyruvate MRSI after systemic delivery of [1-13C] pyruvic acid. Steady-state metabolomic analysis of xenograft tumors was performed with mass spectrometry and steady-state lactate concentrations were measured with proton (1H) MRS. Perfusion and oxygenation of xenografts were measured with electron paramagnetic resonance (EPR) imaging with OX063. Tumor growth was assessed after lactate dehydrogenase (LDH) inhibition with FX-11 (42 μg/mouse/day for 5 days × 2 weekly cycles). Lactate production, pyruvate uptake, extracellular acidification rates, and oxygen consumption of the prostate cancer cell lines were analyzed in vitro. LDH activity was assessed in tumor homogenates. Results: DU145 tumors demonstrated an enhanced conversion of pyruvate to lactate with hyperpolarized [1-13C]-pyruvate MRSI compared with PC3 and a corresponding greater sensitivity to LDH inhibition. No difference was observed between PC3 and DU145 xenografts in steady-state measures of pyruvate fermentation, oxygenation, or perfusion. The two cell lines exhibited similar sensitivity to FX-11 in vitro. LDH activity correlated to FX-11 sensitivity. Conclusions: Hyperpolarized [1-13C]-pyruvate MRSI of prostate cancer predicts efficacy of targeting the Warburg effect. Clin Cancer Res; 24(13); 3137–48. ©2018 AACR.

AIMS: Evofosfamide (TH-302) is a hypoxia-activated prodrug (HAP) that releases the DNA-damaging bromo-isophosphoramide mustard (Br-IPM) moiety selectively under hypoxic conditions. Since solid tumors are known to have hypoxic regions, HAPs in combination with chemotherapy or radiotherapy (XRT) will be beneficial. We tested the oxygen dependence of release kinetics of Br-IPM using electron paramagnetic resonance (EPR) with spin trapping by monitoring redox cycling of the nitroimidazole moiety of TH-302, and oxygen dependence of TH-302 on in vitro cytotoxicity at different levels of hypoxia was also examined. Two tumor implants (SCCVII and HT29) in mice were studied. RESULTS: TH-302 fragmentation to release Br-IPM was noticed at oxygen levels

Nuclear hyperpolarization is a phenomenon that can be used to improve the sensitivity of magnetic resonance molecular sensors. However, such sensors typically suffer from short hyperpolarization lifetime. Herein we report that [15N, D14]trimethylphenylammonium (TMPA) has a remarkably long spin–lattice relaxation time (1128 s, 14.1 T, 30 °C, D2O) on its 15N nuclei and achieves a long retention of the hyperpolarized state. [15N, D14]TMPA-based hyperpolarized sensor for carboxylesterase allowed the highly sensitive analysis of enzymatic reaction by 15N NMR for over 40 min in phophate-buffered saline (H2O, pH 7.4, 37 °C).

The γ-glutamyl transpeptidase (GGT) enzyme plays a central role in glutathione homeostasis. Direct detection of GGT activity could provide critical information for the diagnosis of several pathologies. We propose a new molecular probe, γ-Glu-[1-(13) C]Gly, for monitoring GGT activity in vivo by hyperpolarized (HP) (13) C magnetic resonance (MR). The properties of γ-Glu-[1-(13) C]Gly are suitable for in vivo HP (13) C metabolic analysis since the chemical shift between γ-Glu-[1-(13) C]Gly and its metabolic product, [1-(13) C]Gly, is large (4.3 ppm) and the T1 of both compounds is relatively long (30 s and 45 s, respectively, in H2 O at 9.4 T). We also demonstrate that γ-Glu-[1-(13) C]Gly is highly sensitive to in vivo modulation of GGT activity induced by the inhibitor acivicin.

Etoposide (VP-16) is an anti-tumor compound that targets topoisomerase II (top II). In this study, we have identified an alternative binding protein of etoposide by screening a library of T7 phage-displayed peptides. After four rounds of selection using a biotinylated etoposide derivative immobilized on a streptavidin-coated plate, T7 phage particles that display a 16-mer peptide NSSASSRGNSSSNSVY (ETBP16) or a 10-mer NSLRKYSKLK (ETBP10) were enriched with the ratio of 40 or 11 out of the 69 clones, respectively. Binding of etoposide to these peptides was confirmed by surface plasmon resonance (SPR) analysis, which showed ETBP16 and ETBP10 to have a kinetic constant of 4.85 × 10−5 M or 6.45 × 10−5 M, respectively. ETBP16 displays similarity with the ser-rich domain in E2F-4, a transcription factor in cell cycle-regulated genes, suggesting that etoposide might interact with E2F-4 via this domain. SPR analysis confirmed the specific binding of etoposide to recombinant E2F-4 is in the order of 10−5 M. Furthermore, etoposide was shown to inhibit luciferase reporter gene expression mediated by the heterodimeric E2F-4/DP complex. Taken together, our results suggest that etoposide directly binds to E2F-4 and inhibits subsequent gene transcription mediated by heterodimeric E2F-4/DP complexes in the nucleus.

Beta-sulfoquinovosyldiacylglycerol (betaSQDG) is a synthetic sulfoglycolipid that shows inhibitory activity of DNA polymerase lambda (pol lambda). Here we identified a betaSQDG binding region within murine pol lambda (Mmpol lambda) using T7 phage display technology. We compared the binding intensity of betaSQDG with recombinant phages (phages lambda1-6) that displayed different segments of Mmpol lambda. The binding assay clearly showed that phage lambda1, which displayed the non-structural Met1-Arg95 region including the nuclear localization signal (NLS) and part of the BRCT domain, bound more strongly to betaSQDG than the other recombinant phages. Binding assays using recombinant proteins gave similar results, showing specific betaSQDG binding to Met1-Arg95 with a K(D) value of 9.9 nM. Furthermore, in a cell-based assay, nuclear localization of EGFP-pollambda was inhibited in the presence of betaSQDG possibly due to binding of betaSQDG to NLS. These experiments clearly show that the binding region of betaSQDG within Mmpol lambda could be successfully identified using T7 phage display technology. We suggest that the strategy we describe here will be of value for identifying the binding site within a protein for small ligands, and will provide information that cannot be obtained using other experimental techniques due to their inherent technical limitations.

Purpose: X-ray irradiation of tumors causes diverse effects on the tumor microenvironment, including metabolism. Recent developments of hyperpolarized 13C-MRI enabled detecting metabolic changes in tumors using a tracer [1-13C]pyruvate, which participates in important bioenergetic processes that are altered in cancers. Here, we investigated the effects of X-ray irradiation on pyruvate metabolism in squamous cell carcinoma (SCCVII) and colon cancer (HT-29) using hyperpolarized 13C-MRI. Experimental Design: SCCVII and HT-29 tumors were grown by injecting tumor cells into the hind legs of mice. [1-13C]pyruvate was hyperpolarized and injected intravenously into tumor-bearing mice, and 13C-MR signals were acquired using a 4.7 T scanner. Results: [1-13C]pyruvate and [1-13C]lactate were detected in the tumor-bearing legs immediately after hyperpolarized [1-13C]pyruvate administration. The [1-13C]lactate to [1-13C]pyruvate ratio (Lac/Pyr) increased as the tumors grew in nonirradiated SCCVII tumors. The increase in Lac/Pyr was suppressed modestly with a single 10 Gy of irradiation, but it significantly decreased by further irradiation (10 Gy × 3). Similar results were obtained in HT-29; Lac/Pyr significantly dropped with fractionated 30 Gy irradiation. Independent ex vivo measurements revealed that the lactate dehydrogenase (LDH) activity and protein level were significantly smaller in the irradiated SCCVII tumors compared with the nonirradiated tumors, indicating that a decrease in LDH activity was one of the main factors responsible for the decrease of Lac/Pyr observed on 13C-MRI. Conclusions: Robust changes of Lac/Pyr observed in the HT-29 after the radiation suggested that lactate conversion from pyruvate monitored with hyperpolarized 13C-MRI could be useful for the evaluation of early response to radiotherapy. Clin Cancer Res; 21(22); 5073–81. ©2015 AACR. See related commentary by Lai et al., p. 4996

Aims: The tumor microenvironment is characterized by a highly reducing redox status, a low pH, and hypoxia. Anti-angiogenic therapies for solid tumors frequently function in two steps: the transient normalization of structurally and functionally aberrant tumor blood vessels with increased blood perfusion, followed by the pruning of tumor blood vessels and the resultant cessation of nutrients and oxygen delivery required for tumor growth. Conventional anatomic or vascular imaging is impractical or insufficient to distinguish between the two steps of tumor response to anti-angiogenic therapies. Here, we investigated whether the noninvasive imaging of the tumor redox state and energy metabolism could be used to characterize anti-angiogenic drug-induced transient vascular normalization. Results: Daily treatment of squamous cell carcinoma (SCCVII) tumor-bearing mice with the multi-tyrosine kinase inhibitor sunitinib resulted in a rapid decrease in tumor microvessel density and the suppression of tumor growth. Tumor pO2 imaging by electron paramagnetic resonance imaging showed a transient increase in tumor oxygenation after 2–4 days of sunitinib treatment, implying improved tumor perfusion. During this window of vascular normalization, magnetic resonance imaging of the redox status using an exogenously administered nitroxide probe and hyperpolarized 13C MRI of the metabolic flux of pyruvate/lactate couple revealed an oxidative shift in tumor redox status. Innovation: Redox-sensitive metabolic couples can serve as noninvasive surrogate markers to identify the vascular normalization window in tumors with imaging techniques. Conclusion: A multimodal imaging approach to characterize physiological, metabolic, and redox changes in tumors is useful to distinguish between the different stages of anti-angiogenic treatment. Antioxid. Redox Signal. 21, 1145–1155.

Background Methotrexate (MTX) is an agent used in chemotherapy of tumors and autoimmune disease including rheumatoid arthritis (RA). In addition, MTX has some anti-inflammatory activity. Although dihydrofolate reductase (DHFR) is a well-known target for the anti-tumor effect of MTX, the mode of action for the anti-inflammatory activity of MTX is not fully understood. Methodology/Result Here, we performed a screening of MTX-binding proteins using T7 phage display with a synthetic biotinylated MTX derivative. We then characterized the interactions using surface plasmon resonance (SPR) analysis and electrophoretic mobility shift assay (EMSA). Using a T7 phage display screen, we identified T7 phages that displayed part of high-mobility group box 1 (HMGB1) protein (K86-V175). Binding affinities as well as likely binding sites were characterized using genetically engineered truncated versions of HMGB1 protein (Al G1-K87, Bj: F88-K181), indicating that MTX binds to HMGB1 via two independent sites with a dissociation constants (KD) of 0.50±0.03 µM for Al and 0.24±0.01 µM for Bj. Although MTX did not inhibit the binding of HMGB1 to DNA via these domains, HMGB1/RAGE association was impeded in the presence of MTX. These data suggested that binding of MTX to part of the RAGE-binding region (K149-V175) in HMGB1 might be significant for the anti-inflammatory effect of MTX. Indeed, in murine macrophage-like cells (RAW 264.7), TNF-α release and mitogenic activity elicited by specific RAGE stimulation with a truncated monomeric HMGB1 were inhibited in the presence of MTX. Conclusions/Significance These data demonstrate that HMGB1 is a direct binding protein of MTX. Moreover, binding of MTX to RAGE-binding region in HMGB1 inhibited the HMGB1/RAGE interaction at the molecular and cellular levels. These data might explain the molecular basis underlying the mechanism of action for the anti-inflammatory effect of MTX.

Here, we describe an improved system for protein crystallization based on heterogeneous nucleation using fluorinated layered silicate. In addition, we also investigated the mechanism of nucleation on the silicate surface. Crystallization of lysozyme using silicates with different chemical compositions indicated that fluorosilicates promoted nucleation whereas the silicates without fluorine did not. The use of synthesized saponites for lysozyme crystallization confirmed that the substitution of hydroxyl groups contained in the lamellae structure for fluorine atoms is responsible for the nucleation-inducing property of the nucleant. Crystallization of twelve proteins with a wide range of pI values revealed that the nucleation promoting effect of the saponites tended to increase with increased substitution rate. Furthermore, the saponite with the highest fluorine content promoted nucleation in all the test proteins regardless of their overall net charge. Adsorption experiments of proteins on the saponites confirmed that the density of adsorbed molecules increased according to the substitution rate, thereby explaining the heterogeneous nucleation on the silicate surface.

Two anti-mycobacterial peptides with a lasso structure, named lariatins A and B, were separated by HP-20 and ODS column chromatographies and purified by HPLC from the culture broth of Rhodococcus jostii K01-B0171, which was isolated from soil aggregates collected in Yunnan, China. Lariains A and B showed growth inhibition against Mycobacterium smegmatis with MIC values of 3.13 and 6.25 microg/ml in agar dilution method, respectively. Furthermore, lariatin A inhibited the growth of Mycobacterium tuberculosis with an MIC of 0.39 microg/ml in liquid microdilution method.

The X family DNA polymerases lambda (CcPollambda) and mu (CcPolmu) were shown to be expressed during meiotic prophase in the basidiomycete, Coprinus cinereus. These two polymerases are the only members of the X family in the C. cinereus genome. The open reading frame of CcPollambda encoded a predicted product of 800 amino acid residues and that of CcPolmicro of 621 amino acid residues. Both CcPollambda and CcPolmicro required Mn(2+) ions for activity, and both were strongly inhibited by dideoxythymidine triphosphate. Unlike their mammalian counterparts, CcPollambda and CcPolmicro had no terminal deoxynucleotidyl transferase activity. Immunostaining analysis revealed that CcPollambda was present at meiotic prophase nuclei in zygotene and pachytene cells, which is the period when homologous chromosomes pair and recombine. CcPolmicro was present in a slightly wider range of cell stages, zygotene to diplotene. In analyses using D-loop recombination intermediate substrates, we found that both CcPollambda and CcPolmicro could promote primer extension of an invading strand in a D-loop structure. Moreover, both polymerases could fully extend the primer in the D-loop substrate, suggesting that D-loop extension is an activity intrinsic to CcPollambda and CcPolmicro. Based on these data, we discuss the possible roles of these polymerases in meiosis.

A human replication initiation protein, Cdt1, is a central player in the cell cycle regulation of DNA replication, and geminin down-regulates Cdt1 function by direct binding. It has been demonstrated that Cdt1 hyperfunction resulting from Cdt1-geminin imbalance, for example, by geminin silencing with small interfering RNA, induces DNA re-replication and eventual cell death in some cancer-derived cell lines. We established a high throughput screening system based on a modified enzyme-linked immunosorbent assay to identify compounds that interfere with human Cdt1-geminin binding. Using this system, we screened inhibitors from natural compounds, and found that a fatty acid, linoleic acid (C18:2), from a basidiomycete, inhibited Cdt1-geminin interaction in vitro. Of the commercially purchased linear-chain fatty acids tested, the inhibitory effect of oleic acid (C18:1) was the strongest, with 50% inhibition observed at concentrations of 9.6 microM. Since trans-configuration, the ester form, and the addition of the hydroxyl group of oleic acid had no influence on C18:1 fatty acid derivatives, both parts of a carboxylic acid and an alkyl chain containing cis-type double bonds of fatty acid might be essential for inhibition. Surface plasmon resonance analysis demonstrated that oleic acid was able to bind selectively to Cdt1, but did not interact with geminin. Using a three-dimensional computer modeling analysis, oleic acid was conjectured to interact with the geminin interaction interface on Cdt1, and the carboxyl group of oleic acid was assumed to form hydrogen bonds with the residue of Arg342 of Cdt1. These results suggested that, at least in vitro, oleic acid-containing cell membranes of the lipid bilayer inhibit Cdt1-geminin complex formation by binding to Cdt1 and thereby liberating Cdt1 from inhibition by geminin.