Catalytic Oxidation of Methanol to Formaldehyde Catalyzed by Iron Complex with N3S3-type Tripodal Ligand.

A Fe3+ complex with N3S3-type tripod ligand, 1, reacts with O2 in CH3OH to generate formaldehyde, which has been studied structurally, spectroscopically, and electrochemically. Complex 1 crystallizes as an octahedral structure with crystallographic C3 symmetry around the metal, with Fe-N=2.2917(17) Å and Fe-S=2.3574(6) Å. UV-vis spectrum of 1 in CH3OH under Ar shows an intense band at 572 nm (ϵ 4,100 M-1cm-1), which shifts to 590 nm (ϵ 2,860 M-1cm-1) by the addition of O2, and a new peak appeared at 781 nm (ϵ 790 M-1cm-1). Such a spectral change is not observed in CH2Cl2. Cyclic voltammogram (CV) of 1 in CH2Cl2 under Ar gives reversible redox waves assigned to Fe2+/Fe3+ and Fe3+/Fe4+ couples at -1.60 V (ΔE=69 mV) and -0.53 V (ΔE=71 mV) vs Fc/Fc+, respectively. In contrast, in CH3OH, the reversible redox waves, albeit accompanied by a positive shift of the Fe2+/Fe3+ couple, are observed at -1.20 V (ΔE=85 mV) and -0.53 V (ΔE=64 mV) vs Fc/Fc+ under Ar. Interestingly, a catalytic current was observed for the CV of 1 in CH3OH in the presence of CH3ONa under Ar, when the sweep rate was slowed down.

Neuroproteomic mapping of kinases and their substrates downstream of acetylcholine: finding and implications.

INTRODUCTION: Since the emergence of the cholinergic hypothesis of Alzheimer's disease (AD), acetylcholine has been viewed as a mediator of learning and memory. Donepezil improves AD-associated learning deficits and memory loss by recovering brain acetylcholine levels. However, it is associated with side effects due to global activation of acetylcholine receptors. Muscarinic acetylcholine receptor M1 (M1R), a key mediator of learning and memory, has been an alternative target. The importance of targeting a specific pathway downstream of M1R has recently been recognized. Elucidating signaling pathways beyond M1R that lead to learning and memory holds important clues for AD therapeutic strategies. AREAS COVERED: This review first summarizes the role of acetylcholine in aversive learning, one of the outputs used for preliminary AD drug screening. It then describes the phosphoproteomic approach focused on identifying acetylcholine intracellular signaling pathways leading to aversive learning. Finally, the intracellular mechanism of donepezil and its effect on learning and memory is discussed. EXPERT OPINION: The elucidation of signaling pathways beyond M1R by phosphoproteomic approach offers a platform for understanding the intracellular mechanism of AD drugs and for developing AD therapeutic strategies. Clarifying the molecular mechanism that links the identified acetylcholine signaling to AD pathophysiology will advance the development of AD therapeutic strategies.

Preparations of trans- and cis-µ-1,2-Peroxodiiron(III) Complexes.

The iron(II) complex with cis,cis-1,3,5-tris(benzylamino)cyclohexane (Bn3CY) (1) has been synthesized and characterized, which reacted with dioxygen to form the peroxo complex 2 in acetone at -60 °C. On the basis of spectroscopic measurements for 2, it was confirmed that the peroxo complex 2 has a trans-µ-1,2 fashion. Additionally, the peroxo complex 2 was reacted with benzoate anion as a bridging agent to give a peroxo complex 3. The results of resonance Raman and 1H-NMR studies supported that the peroxo complex 3 is a cis-µ-1,2-peroxodiiron(III) complex. These spectral features were interpreted by using DFT calculations.

Extracellular and Intracellular Signaling for Neuronal Polarity.

Neurons are one of the highly polarized cells in the body. One of the fundamental issues in neuroscience is how neurons establish their polarity; therefore, this issue fascinates many scientists. Cultured neurons are useful tools for analyzing the mechanisms of neuronal polarization, and indeed, most of the molecules important in their polarization were identified using culture systems. However, we now know that the process of neuronal polarization in vivo differs in some respects from that in cultured neurons. One of the major differences is their surrounding microenvironment; neurons in vivo can be influenced by extrinsic factors from the microenvironment. Therefore, a major question remains: How are neurons polarized in vivo? Here, we begin by reviewing the process of neuronal polarization in culture conditions and in vivo. We also survey the molecular mechanisms underlying neuronal polarization. Finally, we introduce the theoretical basis of neuronal polarization and the possible involvement of neuronal polarity in disease and traumatic brain injury.

Muscarinic signaling regulates voltage-gated potassium channel KCNQ2 phosphorylation in the nucleus accumbens via protein kinase C for aversive learning.

The nucleus accumbens (NAc) plays critical roles in emotional behaviors, including aversive learning. Aversive stimuli such as an electric foot shock increase acetylcholine (ACh) in the NAc, and muscarinic signaling appears to increase neuronal excitability and aversive learning. Muscarinic signaling inhibits the voltage-dependent potassium KCNQ current which regulates neuronal excitability, but the regulatory mechanism has not been fully elucidated. Phosphorylation of KCNQ2 at threonine 217 (T217) and its inhibitory effect on channel activity were predicted. However, whether and how muscarinic signaling phosphorylates KCNQ2 in vivo remains unclear. Here, we found that PKC directly phosphorylated KCNQ2 at T217 in vitro. Carbachol and a muscarinic M1 receptor (M1R) agonist facilitated KCNQ2 phosphorylation at T217 in NAc/striatum slices in a PKC-dependent manner. Systemic administration of the cholinesterase inhibitor donepezil, which is commonly used to treat dementia, and electric foot shock to mice induced the phosphorylation of KCNQ2 at T217 in the NAc, whereas phosphorylation was suppressed by an M1R antagonist. Conditional deletion of Kcnq2 in the NAc enhanced electric foot shock induced aversive learning. Our findings indicate that muscarinic signaling induces the phosphorylation of KCNQ2 at T217 via PKC activation for aversive learning.

New Cluster Analysis Method for Quantitative Dynamic Contrast-Enhanced MRI Assessing Tumor Heterogeneity Induced by a Tumor-Microenvironmental Ameliorator (E7130) Treatment to a Breast Cancer Mouse Model.

BACKGROUND: Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) can provide insight into tumor perfusion. However, a method that can quantitatively measure the intra-tumor distribution of tumor voxel clusters with a distinct range of Ktrans and ve values remains insufficiently explored. HYPOTHESIS: Two-dimensional cluster analysis may quantify the distribution of a tumor voxel subregion with a distinct range of Ktrans and ve values in human breast cancer xenografts. STUDY TYPE: Prospective longitudinal study. ANIMAL MODEL: Twenty-two female athymic nude mice with MCF-7 xenograft, treated with E7130, a tumor-microenvironmental ameliorator, or saline. FIELD STRENGTH/SEQUENCE: 9.4 Tesla, turbo rapid acquisition with relaxation enhancement, and spoiled gradient-echo sequences. ASSESSMENT: We performed two-dimensional k-means clustering to identify tumor voxel clusters with a distinct range of Ktrans and ve values on Days 0, 2, and 5 after treatment, calculated the ratio of the number of tumor voxels in each cluster to the total number of tumor voxels, and measured the normalized distances defined as the ratio of the distance between each tumor voxel and the nearest tumor margin to a tumor radius. STATISTICAL TESTS: Unpaired t-tests, Dunnett's multiple comparison tests, and Chi-squared test were used. RESULTS: The largest and second largest clusters constituted 44.4% and 27.5% of all tumor voxels with cluster centroid values of Ktrans at 0.040 min-1 and 0.116 min-1 , and ve at 0.131 and 0.201, respectively. At baseline (Day 0), the average normalized distances for the largest and second largest clusters were 0.33 and 0.24, respectively. E7130-treated group showed the normalized distance of the initial largest cluster decreasing to 0.25, while that of the second largest cluster increasing to 0.31. Saline-treated group showed no change. DATA CONCLUSION: A two-dimensional cluster analysis might quantify the spatial distribution of a tumor subregion with a distinct range of Ktrans and ve values. LEVEL OF EVIDENCE: 1 TECHNICAL EFFICACY: Stage 1.

Rho-Rho-Kinase Regulates Ras-ERK Signaling Through SynGAP1 for Dendritic Spine Morphology.

The structural plasticity of dendritic spines plays a critical role in NMDA-induced long-term potentiation (LTP) in the brain. The small GTPases RhoA and Ras are considered key regulators of spine morphology and enlargement. However, the regulatory interaction between RhoA and Ras underlying NMDA-induced spine enlargement is largely unknown. In this study, we found that Rho-kinase/ROCK, an effector of RhoA, phosphorylated SynGAP1 (a synaptic Ras-GTPase activating protein) at Ser842 and increased its interaction with 14-3-3ζ, thereby activating Ras-ERK signaling in a reconstitution system in HeLa cells. We also found that the stimulation of NMDA receptor by glycine treatment for LTP induction stimulated SynGAP1 phosphorylation, Ras-ERK activation, spine enlargement and SynGAP1 delocalization from the spines in striatal neurons, and these effects were prevented by Rho-kinase inhibition. Rho-kinase-mediated phosphorylation of SynGAP1 appeared to increase its dissociation from PSD95, a postsynaptic scaffolding protein located at postsynaptic density, by forming a complex with 14-3-3ζ. These results suggest that Rho-kinase phosphorylates SynGAP1 at Ser842, thereby activating the Ras-ERK pathway for NMDA-induced morphological changes in dendritic spines.

Distinctive Aspects in Aquation, Proton-Coupled Redox, and Photoisomerization Reactions between Geometric Isomers of Mononuclear Ruthenium Complexes with a Large-π-Conjugated Tetradentate Ligand.

Geometric isomers of mononuclear ruthenium(II) complexes, distal-/proximal-[Ru(tpy)(dpda)Cl]+ (d-/p-RuCl, tpy = 2,2':6',2″-terpyridine, dpda = 2,7-bis(2-pyridyl)-1,8-diazaanthracene), were newly synthesized to comprehensively investigate the geometric and electronic structures and distinctive aspects in various reactions between isomers. The ultraviolet (UV)-visible absorption spectra of d-/p-RuCl isomers show intense bands for metal-to-ligand charge transfer (MLCT) at close wavelengths of 576 and 573 nm, respectively. However, time-dependent density functional theory (TD-DFT) calculations suggest that the MLCT transition of d-RuCl involves mainly single transitions to the π* orbital of the dpda ligand in contrast to mixing of the π* orbitals of the dpda and tpy ligands for p-RuCl. The aquation reaction (1.5 × 10-3 s-1) of p-RuCl to yield proximal-[Ru(tpy)(dpda)(OH2)]2+ (p-RuH2O) is faster than that (5.3 × 10-6 s-1) of d-RuCl in D2O/CD3OD (4:1 v/v) by three orders of magnitude, which resulted from the longer Ru-Cl bond by 0.017 Å and the distorted angle (100.2(3)°) of Cl-Ru-N (a nitrogen of dpda, being on a tpy plane) due to the steric repulsion between Cl and dpda for p-RuCl. Electrochemical measurements showed that d-RuH2O undergoes a 2-step oxidation reaction of 1H+-coupled 1e- processes of RuII-OH2/RuIII-OH and RuIII-OH/RuIV═O at pH 1-9, whereas p-RuH2O undergoes a 1-step oxidation reaction of a 2H+-coupled 2e- process of RuII-OH2/RuIV═O in the pH range of pH 1-10. The irreversible photoisomerization from d-RuH2O to p-RuH2O was observed in aqueous solution with an internal quantum yield (Φ) of 5.4 × 10-3% at 520 nm, which is lower compared with Φ = 1.1-2.1% of mononuclear Ru(II) aquo complexes with similar bidentate ligands instead of dpda by three orders of magnitude. This is possibly ascribed to the faster nonradiative decay rate from the excited 3MLCT state to the ground state for d-RuH2O due to the lower π* level of dpda ligands according to the energy-gap law: the rate decreases exponentially with the increasing energy gap.

Phosphorylation Signals Downstream of Dopamine Receptors in Emotional Behaviors: Association with Preference and Avoidance.

Dopamine regulates emotional behaviors, including rewarding and aversive behaviors, through the mesolimbic dopaminergic pathway, which projects dopamine neurons from the ventral tegmental area to the nucleus accumbens (NAc). Protein phosphorylation is critical for intracellular signaling pathways and physiological functions, which are regulated by neurotransmitters in the brain. Previous studies have demonstrated that dopamine stimulated the phosphorylation of intracellular substrates, such as receptors, ion channels, and transcription factors, to regulate neuronal excitability and synaptic plasticity through dopamine receptors. We also established a novel database called KANPHOS that provides information on phosphorylation signals downstream of monoamines identified by our kinase substrate screening methods, including dopamine, in addition to those reported in the literature. Recent advances in proteomics techniques have enabled us to clarify the mechanisms through which dopamine controls rewarding and aversive behaviors through signal pathways in the NAc. In this review, we discuss the intracellular phosphorylation signals regulated by dopamine in these two emotional behaviors.

Rho-Kinase/ROCK Phosphorylates PSD-93 Downstream of NMDARs to Orchestrate Synaptic Plasticity.

The N-methyl-D-aspartate receptor (NMDAR)-mediated structural plasticity of dendritic spines plays an important role in synaptic transmission in the brain during learning and memory formation. The Rho family of small GTPase RhoA and its downstream effector Rho-kinase/ROCK are considered as one of the major regulators of synaptic plasticity and dendritic spine formation, including long-term potentiation (LTP). However, the mechanism by which Rho-kinase regulates synaptic plasticity is not yet fully understood. Here, we found that Rho-kinase directly phosphorylated discs large MAGUK scaffold protein 2 (DLG2/PSD-93), a major postsynaptic scaffold protein that connects postsynaptic proteins with NMDARs; an ionotropic glutamate receptor, which plays a critical role in synaptic plasticity. Stimulation of striatal slices with an NMDAR agonist induced Rho-kinase-mediated phosphorylation of PSD-93 at Thr612. We also identified PSD-93-interacting proteins, including DLG4 (PSD-95), NMDARs, synaptic Ras GTPase-activating protein 1 (SynGAP1), ADAM metallopeptidase domain 22 (ADAM22), and leucine-rich glioma-inactivated 1 (LGI1), by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Among them, Rho-kinase increased the binding of PSD-93 to PSD-95 and NMDARs. Furthermore, we found that chemical-LTP induced by glycine, which activates NMDARs, increased PSD-93 phosphorylation at Thr612, spine size, and PSD-93 colocalization with PSD-95, while these events were blocked by pretreatment with a Rho-kinase inhibitor. These results indicate that Rho-kinase phosphorylates PSD-93 downstream of NMDARs, and suggest that Rho-kinase mediated phosphorylation of PSD-93 increases the association with PSD-95 and NMDARs to regulate structural synaptic plasticity.

Dynamic subcellular localization and transcription activity of the SRF cofactor MKL2 in the striatum are regulated by MAPK.

Dopamine type 1 receptor (D1R) signaling activates protein kinase A (PKA), which then activates mitogen-activated protein kinase (MAPK) through Rap1, in striatal medium spiny neurons (MSNs). MAPK plays a pivotal role in reward-related behavior through the activation of certain transcription factors. How D1R signaling regulates behavior through transcription factors remains largely unknown. CREB-binding protein (CBP) promotes transcription through hundreds of different transcription factors and is also important for reward-related behavior. To identify transcription factors regulated by dopamine signaling in MSNs, we performed a phosphoproteomic analysis using affinity beads coated with CBP. We obtained approximately 40 novel candidate proteins in the striatum of the C57BL/6 mouse brain after cocaine administration. Among them, the megakaryoblastic leukemia-2 (MKL2) protein, a transcriptional coactivator of serum response factor (SRF), was our focus. We found that the interaction between CBP and MKL2 was increased by cocaine administration. Additionally, MKL2, CBP and SRF formed a ternary complex in vivo. The C-terminal domain of MKL2 interacted with CBP-KIX and was phosphorylated by MAPK in COS7 cells. The activation of PKA-MAPK signaling induced the nuclear localization of MKL2 and increased SRF-dependent transcriptional activity in neurons. These results demonstrate that dopamine signaling regulates the interaction of MKL2 with CBP in a phosphorylation-dependent manner and thereby controls SRF-dependent gene expression. Cover Image for this issue: https://doi.org/10.1111/jnc.15067.

Correlative serum biomarker analyses in the phase 2 trial of lenvatinib-plus-everolimus in patients with metastatic renal cell carcinoma.

BACKGROUND: No biomarkers have been established to predict treatment efficacy in renal cell carcinoma (RCC). In an exploratory retrospective analysis of a Phase 2 study, we constructed composite biomarker scores (CBSs) to predict progression-free survival (PFS) and overall survival (OS) in patients with metastatic RCC randomised to receive lenvatinib-plus-everolimus. METHODS: Of 40 biomarkers tested, the 5 most strongly associated with PFS (HGF, MIG, IL-18BP, IL-18, ANG-2) or OS (TIMP-1, M-CSF, IL-18BP, ANG-2, VEGF) were used to make a 5-factor PFS-CBS or OS-CBS, respectively. A 2-factor CBS was generated with biomarkers common to PFS-CBS and OS-CBS. Patients were divided into groups accordingly (5-factor-CBS high: 3-5, CBS-low: 0-2; 2-factor-CBS high: 1-2, CBS-low: 0). RESULTS: PFS/OS with lenvatinib-plus-everolimus were significantly longer in the 5-factor CBS-high group versus the CBS-low group (P = 0.0022/P < 0.0001, respectively). In the CBS-high group, PFS/OS were significantly longer with lenvatinib-plus-everolimus versus everolimus (P < 0.001/P = 0.0079, respectively); PFS was also significantly longer with lenvatinib-plus-everolimus versus lenvatinib (P = 0.0046). The 5-factor-CBS had a predictive role in PFS and OS after multivariate analysis. Similar trends were observed with the 2-factor-CBS for PFS (i.e., lenvatinib-plus-everolimus versus everolimus). CONCLUSIONS: The 5-factor CBS may identify patients with metastatic RCC who would benefit from lenvatinib-plus-everolimus versus everolimus; additional validation is required. CLINICAL TRIAL REGISTRATION: The clinical trial registration number is NCT01136733.

The LEAP program: lenvatinib plus pembrolizumab for the treatment of advanced solid tumors.

Tumor progression and immune evasion result from multiple oncogenic and immunosuppressive signals within the tumor microenvironment. The combined blockade of VEGF and inhibitory immune checkpoint signaling has been shown to enhance immune activation and tumor destruction in preclinical models. The LEAP clinical trial program is evaluating the safety and efficacy of lenvatinib (a multikinase inhibitor) plus pembrolizumab (a PD-1 inhibitor) across several solid tumor types. Preliminary results from ongoing trials demonstrate robust antitumor activity and durable responses across diverse tumor types with a manageable safety profile. Thus, lenvatinib plus pembrolizumab is anticipated to be an important potential new regimen for several solid cancers that currently have limited therapeutic options. Clinical trial registration: NCT03884101, NCT03713593, NCT03820986, NCT03776136, NCT03797326, NCT03829319, NCT03829332, NCT03976375, NCT04428151, NCT04199104, NCT03898180, NCT04246177 (ClinicalTrials.gov).

Second-line lenvatinib in patients with recurrent endometrial cancer.

OBJECTIVE: This study assessed the efficacy of lenvatinib, a multitargeted tyrosine kinase inhibitor, as second-line therapy in patients with unresectable endometrial cancer. The primary end point was the objective response rate (ORR) as assessed by independent radiologic review (IRR). Secondary end points included median progression-free survival (PFS), overall survival (OS), and clinical benefit rate. Exploratory end points examined the association of baseline levels of plasma biomarkers (50 circulating cytokine and/or angiogenic factors measured by immunoassays) with efficacy outcomes. METHODS: An international, open-label, single-arm, multicenter, phase 2 trial was conducted. Eligible patients had histologically confirmed unresectable endometrial cancer that relapsed after 1 prior systemic platinum-based chemotherapy. Patients received once-daily oral lenvatinib 24 mg in a 28-day dosing cycle. RESULTS: There were 133 patients in the study. By IRR, 19 patients had a confirmed objective response for an ORR of 14.3% (95% CI: 8.8-21.4). Durable stable disease (≥23 weeks) was observed in 31 patients (23.3%) and the clinical benefit rate was 37.6% (95% CI: 29.3-46.4). Median PFS was 5.6 months (95% CI: 3.7-6.3), and median OS was 10.6 months (95% CI: 8.9-14.9). The most common (any grade) treatment-related adverse events were fatigue/asthenia (48%), hypertension (49%), nausea/vomiting (32%), decreased appetite (32%), and diarrhea (31%). Lower baseline levels of angiopoietin-2 were associated with longer PFS, OS, and a higher ORR. CONCLUSIONS: Patients with recurrent endometrial cancer treated with second-line lenvatinib experienced modest antitumor activity and treatment was generally well tolerated, with a safety profile consistent with previous studies.

Lenvatinib induces death of human hepatocellular carcinoma cells harboring an activated FGF signaling pathway through inhibition of FGFR-MAPK cascades.

Lenvatinib inhibits VEGF- and FGF-driven angiogenesis, and proliferation of tumor cells with activated FGF signaling pathways in preclinical models, and we previously demonstrated antitumor activity in human HCC xenograft tumor models. Here, we examined the inhibitory activity of lenvatinib against FGF-driven survival of human HCC cell lines. First, we conducted a histological analysis of FGF19-overexpressing Hep3B2.1-7 xenograft tumors collected from mice treated with lenvatinib. Second, we examined the effects of pharmacological inhibition on survival of cultured HCC cells with an activated FGF signaling pathway under nutrient-starved culture condition to mimic tumor microenvironments induced by angiogenesis inhibition. In the first analysis, area of histological focal necrosis was greater in Hep3B2.1-7 xenograft tumors with the lenvatinib treatment than that after the treatment with sorafenib, which does not inhibit FGFRs. Lenvatinib and E7090 (a selective FGFR1-3 inhibitor), but not sorafenib, induced death of Hep3B2.1-7, and another FGF19 overexpressing HuH-7 cells. Lenvatinib and E7090 decreased phosphorylation of downstream molecules of the FGF signaling pathway (such as FRS2, Erk, and p38 MAPK), and induced PARP cleavage, even under limited nutrients. PD0325901, MEK inhibitor, caused the same changes in HCC cells as those described above for lenvatinib and E7090. These results reveal that the FGF signaling pathway through MAPK cascades plays an important role in survival of HCC cell lines with an activated FGF signaling pathway under limited nutrients, and FGFR-MAPK cascades likely contribute to survival of HCC cells with an activated FGF signaling pathway under tumor microenvironments with limited nutrients, where tumor angiogenesis is inhibited.

Antitumor and Antiangiogenic Activities of Lenvatinib in Mouse Xenograft Models of Vascular Endothelial Growth Factor-Induced Hypervascular Human Hepatocellular Carcinoma.

High expression of vascular endothelial growth factor (VEGF) in patients with hepatocellular carcinoma (HCC) is associated with poor prognosis. Here, we investigated the antitumor activity of lenvatinib, a multiple receptor tyrosine kinase inhibitor, in VEGF-overexpressing HCC models. In human umbilical vein endothelial cells, lenvatinib showed potent inhibitory activities against VEGF-induced proliferation and VEGF/basic fibroblast growth factor-induced tube formation. In VEGF-overexpressing HCC xenograft models, characterized by aggressive tumor growth and hypervascularity, lenvatinib had significant antitumor and antiangiogenic activities. These results suggest that potent activity of lenvatinib against VEGF signaling underlies its antitumor and antiangiogenic activities in the hypervascular HCC models.

Immunomodulatory activity of lenvatinib contributes to antitumor activity in the Hepa1-6 hepatocellular carcinoma model.

Angiogenesis inhibitors such as lenvatinib and sorafenib, and an immune checkpoint inhibitor (ICI), nivolumab, are used for anticancer therapies against advanced hepatocellular carcinoma (HCC). Combination treatments comprising angiogenesis inhibitors plus ICIs are promising options for improving clinical benefits in HCC patients, and clinical trials are ongoing. Here, we investigated the antitumor and immunomodulatory activities of lenvatinib (a multiple receptor tyrosine kinase inhibitor targeting vascular endothelial growth factor receptor 1-3, fibroblast growth factor receptor 1-4, platelet-derived growth factor receptor α, KIT and RET) and the combined antitumor activity of lenvatinib plus anti-programmed cell death 1 (PD-1) antibody in the Hepa1-6 mouse HCC syngeneic model. We found that the antitumor activities of lenvatinib and sorafenib were not different in immunodeficient mice, but lenvatinib showed more potent antitumor activity than sorafenib in immunocompetent mice. The antitumor activity of lenvatinib was greater in immunocompetent mice than in immunodeficient mice and was attenuated by CD8+ T cell depletion. Treatment with lenvatinib plus anti-PD-1 antibody resulted in more tumor regression and a higher response rate compared with either treatment alone in immunocompetent mice. Single-cell RNA sequencing analysis demonstrated that treatment with lenvatinib with or without anti-PD-1 antibody decreased the proportion of monocytes and macrophages population and increased that of CD8+ T cell populations. These data suggest that lenvatinib has immunomodulatory activity that contributes to the antitumor activity of lenvatinib and enhances the antitumor activity in combination treatment with anti-PD-1 antibody. Combination treatment of lenvatinib plus anti-PD-1 antibody therefore warrants further investigation against advanced HCC.

Neuronal polarization.

Neurons are highly polarized cells with structurally and functionally distinct processes called axons and dendrites. This polarization underlies the directional flow of information in the central nervous system, so the establishment and maintenance of neuronal polarization is crucial for correct development and function. Great progress in our understanding of how neurons establish their polarity has been made through the use of cultured hippocampal neurons, while recent technological advances have enabled in vivo analysis of axon specification and elongation. This short review and accompanying poster highlight recent advances in this fascinating field, with an emphasis on the signaling mechanisms underlying axon and dendrite specification in vitro and in vivo.

Co(III) Complexes with N2S3-Type Ligands as Structural/Functional Models for the Isocyanide Hydrolysis Reaction Catalyzed by Nitrile Hydratase.

It has been before reported that, in addition to hydration of nitriles, the Fe-type nitrile hydratase (NHase) also catalyzes the hydrolysis of tert-butylisocyanide ( tBuNC). In order to investigate the unique isocyanide hydrolysis by NHase, we prepared three related Co(III) model complexes, PPh4[Co(L)] (1), PPh4[Co(L-O3)] (2), and PPh4[Co(L-O4)] (3), where L is bis( N-(2-mercapto-2-methylpropionyl)aminopropyl)sulfide. The suffixes L-O3 and L-O4 indicate ligands with a sulfenate and a sulfinate and with two sulfinates, respectively, instead of the two thiolates of L. The X-ray analyses of 1 and 3 reveal trigonal bipyramidal and square pyramidal structures, respectively. Complex 2, however, has five-coordinate trigonal-bipyramidal geometry with η2-type S-O coordination by a sulfenyl group. Addition of tBuNC to 1, 2, and 3 induces an absorption spectral change as a result of formation of an octahedral Co(III) complex. This interpretation is also supported by the crystal structures of PPh4[Co(L-O4)( tBuNC)] (4) and (PPh4)2[Co(L-O4)(CN)] (5). A water molecule interacts with 3 but cannot be activated as reported previously, as demonstrated by the lack of absorption spectral change in the pH range of 5.5-10.2. Interestingly, the coordinated tBuNC is hydrolyzed by 2 and 3 at pH 10.2 to produce tBuNH2 and CO molecule, but 1 does not react. These findings provide strong evidence that hydrolysis of tBuNC by NHase proceeds not by activation of the coordinated water molecule but by coordination of the substrate. The mechanism of the hydrolysis reaction of tBuNC is explained with support provided by DFT calculations; a positively polarized C atom of tBuNC on the Co(III) center is nucleophilically attacked by a hydroxide anion activated through an interaction of the sulfenyl/sulfinyl oxygen with the nucleophile.

Targeting of tumor growth and angiogenesis underlies the enhanced antitumor activity of lenvatinib in combination with everolimus.

The combination of lenvatinib, a multiple receptor tyrosine kinase inhibitor, plus everolimus, a mammalian target of rapamycin (mTOR) inhibitor, significantly improved clinical outcomes versus everolimus monotherapy in a phase II clinical study of metastatic renal cell carcinoma (RCC). We investigated potential mechanisms underlying the antitumor activity of the combination treatment in preclinical RCC models. Lenvatinib plus everolimus showed greater antitumor activity than either monotherapy in three human RCC xenograft mouse models (A-498, Caki-1, and Caki-2). In particular, the combination led to tumor regression in the A-498 and Caki-1 models. In the A-498 model, everolimus showed antiproliferative activity, whereas lenvatinib showed anti-angiogenic effects. The anti-angiogenic activity was potentiated by the lenvatinib plus everolimus combination in Caki-1 xenografts, in which fibroblast growth factor (FGF)-driven angiogenesis may contribute to tumor growth. The combination showed mostly additive activity in vascular endothelial growth factor (VEGF)-activated, and synergistic activity against FGF-activated endothelial cells, in cell proliferation and tube formation assays, as well as strongly suppressed mTOR-S6K-S6 signaling. Enhanced antitumor activities of the combination versus each monotherapy were also observed in mice bearing human pancreatic KP-1 xenografts overexpressing VEGF or FGF. Our results indicated that simultaneous targeting of tumor cell growth and angiogenesis by lenvatinib plus everolimus resulted in enhanced antitumor activity. The enhanced inhibition of both VEGF and FGF signaling pathways by the combination underlies its superior anti-angiogenic activity in human RCC xenograft models.