Protocol for the comprehensive biochemical and cellular profiling of small-molecule degraders using CoraFluor TR-FRET technology.

Comprehensive characterization of small-molecule degraders, including binary and ternary complex formation and degradation efficiency, is critical for bifunctional ligand development and understanding structure-activity relationships. Here, we present a protocol for the biochemical and cellular profiling of small-molecule degraders based on CoraFluor time-resolved fluorescence resonance energy transfer (TR-FRET) technology. We describe steps for labeling antibodies and proteins, tracer saturation binding, binary target engagement, ternary complex profiling, and off-rate determination. We then detail procedures for the quantification of endogenous and GFP fusion proteins in cell lysates. For complete details on the use and execution of this protocol, please refer to Ichikawa et al.1.

The cyclimids: Degron-inspired cereblon binders for targeted protein degradation.

Cereblon (CRBN) is an E3 ligase substrate adapter widely exploited for targeted protein degradation (TPD) strategies. However, achieving efficient and selective target degradation is a preeminent challenge with ligands that engage CRBN. Here, we report that the cyclimids, ligands derived from the C-terminal cyclic imide degrons of CRBN, exhibit distinct modes of interaction with CRBN and offer a facile approach for developing potent and selective bifunctional degraders. Quantitative TR-FRET-based characterization of 60 cyclimid degraders in binary and ternary complexes across different substrates revealed that ternary complex binding affinities correlated strongly with cellular degradation efficiency. Our studies establish the unique properties of the cyclimids as versatile warheads in TPD and a systematic biochemical approach for quantifying ternary complex formation to predict their cellular degradation activity, which together will accelerate the development of ligands that engage CRBN.

The E3 ligase adapter cereblon targets the C-terminal cyclic imide degron.

The ubiquitin E3 ligase substrate adapter cereblon (CRBN) is a target of thalidomide and lenalidomide1, therapeutic agents used in the treatment of haematopoietic malignancies2-4 and as ligands for targeted protein degradation5-7. These agents are proposed to mimic a naturally occurring degron; however, the structural motif recognized by the thalidomide-binding domain of CRBN remains unknown. Here we report that C-terminal cyclic imides, post-translational modifications that arise from intramolecular cyclization of glutamine or asparagine residues, are physiological degrons on substrates for CRBN. Dipeptides bearing the C-terminal cyclic imide degron substitute for thalidomide when embedded within bifunctional chemical degraders. Addition of the degron to the C terminus of proteins induces CRBN-dependent ubiquitination and degradation in vitro and in cells. C-terminal cyclic imides form adventitiously on physiologically relevant timescales throughout the human proteome to afford a degron that is endogenously recognized and removed by CRBN. The discovery of the C-terminal cyclic imide degron defines a regulatory process that may affect the physiological function and therapeutic engagement of CRBN.

Progranulin and Its Receptor Predict Kidney Function Decline in Patients With Type 2 Diabetes.

Progranulin (PGRN), a growth factor, is abundantly expressed in a broad range of tissues and cell types with pleiotropic functions including inflammation, neurodegeneration, and facilitating lysosome acidification. PGRN binds to TNF receptors (TNFR) and inhibits downstream inflammatory signaling pathways. TNFR is a well-known predictor of glomerular filtration rate (GFR) decline in a variety of diseases. Therefore, we measured circulating PGRN in addition to TNFR using an enzyme-linked immunosorbent assay and explored whether it predicted renal prognosis in 201 Japanese patients with type 2 diabetes. During a median follow-up of 7.6 years, 21 participants reached primary renal endpoint, which involves a decline of at least 57% in eGFR from baseline, or the onset of end-stage renal disease. Univariate Cox regression analysis revealed that classical renal measures (GFR and albuminuria), two TNF-related biomarkers (PGRN and TNFR), and BMI were associated with this outcome. Multivariate analysis demonstrated that high levels of PGRN [HR 2.50 (95%CI 2.47-2.52)] or TNFR1 [HR 5.38 (95%CI 5.26-5.50)] were associated with this outcome after adjusting for relevant covariates. The high levels of PGRN as well as TNFR1 were associated with a risk of primary renal outcome in patients with type 2 diabetes after adjusting for established risk factors.

Differential organ-specific inflammatory response to progranulin in high-fat diet-fed mice.

Progranulin (PGRN) has been reported to bind tumor necrosis factor (TNF) receptor and to inhibit TNFα signaling. We evaluated the effect of augmentation of TNFα signaling by PGRN deficiency on the progression of kidney injury. Eight-week-old PGRN knockout (KO) and wild-type (WT) mice were fed a standard diet or high-fat diet (HFD) for 12 weeks. Albuminuria, markers of tubular damage, and renal mRNA levels of inflammatory cytokines were higher in HFD-fed KO (KO-HFD) mice than in HFD-fed WT (WT-HFD) mice. Body weight, vacuolization in proximal tubules, and systemic and adipose tissue inflammatory markers were lower in the KO-HFD mice than in the WT-HFD mice. The renal megalin expression was lower in the KO mice than in the WT mice regardless of the diet type. The megalin expression was also reduced in mouse proximal tubule epithelial cells stimulated with TNFα and in those with PGRN knockdown by small interfering RNA in vitro. PGRN deficiency was associated with both exacerbated renal inflammation and decreased systemic inflammation, including that in the adipose tissue of mice with HFD-induced obesity. Improved tubular vacuolization in the KO-HFD mice might partially be explained by the decreased expression of megalin in proximal tubules.

Effects of L-Carnitine Supplementation in Patients Receiving Hemodialysis or Peritoneal Dialysis.

L-carnitine is an important factor in fatty acid metabolism, and carnitine deficiency is common in dialysis patients. This study evaluated whether L-carnitine supplementation improved muscle spasm, cardiac function, and renal anemia in dialysis patients. Eighty Japanese outpatients (62 hemodialysis (HD) patients and 18 peritoneal dialysis (PD) patients) received oral L-carnitine (600 mg/day) for 12 months; the HD patients further received intravenous L-carnitine injections (1000 mg three times/week) for 12 months, amounting to 24 months of treatment. Muscle spasm incidence was assessed using a questionnaire, and cardiac function was assessed using echocardiography. Baseline free carnitine concentrations were relatively low in patients who underwent dialysis for >4 years. Total carnitine serum concentration, free carnitine, and acylcarnitine significantly increased after oral L-carnitine treatment for 12 months, and after intravenous L-carnitine injection. There was no significant improvement in muscle spasms, although decreased muscle cramping after L-carnitine treatment was reported by 31% of patients who had undergone HD for >4 years. Hemoglobin concentrations increased significantly at 12 and 24 months in the HD group. Therefore, L-carnitine may be effective for reducing muscle cramping and improving hemoglobin levels in dialysis patients, especially those who have been undergoing dialysis for >4 years.

The sodium-glucose cotransporter 2 inhibitor tofogliflozin prevents diabetic kidney disease progression in type 2 diabetic mice.

Trials on cardiovascular and renal outcomes in patients with type 2 diabetes have consistently demonstrated that sodium-glucose cotransporter 2 (SGLT2) inhibitors reduce the risk of diabetic kidney disease (DKD) progression. However, their renal protective mechanisms have yet to be completely understood and the effect on albuminuria reduction in animal models is controversial. We investigated these issues using KK and KK-Ay mice as a control (CTRL) and as a model for type 2 diabetes (DKD), respectively. KK-Ay mice were treated with 0.015% tofogliflozin, which is an SGLT2 inhibitor, starting at seven weeks of age for eight weeks. Compared with the CTRL mice, the DKD mice had higher HbA1c levels and albuminuria. Although tofogliflozin treatment significantly lowered HbA1c levels, it did not reverse albuminuria. Tofogliflozin treatment enhanced damage in both the glomerular (i.e., enlarged mesangial area, increased foot process effacement rate, and decreased number of WT-1-positive cells) and tubulointerstitial (increased protein levels of KIM-1 and MCP-1, increased number of macrophages, and abnormal mitochondrial morphology) areas. Our results suggest that tofogliflozin may prevent glomerular and tubulointerstitial damage, partly by ameliorating hyperglycemia, renal inflammation, and abnormal mitochondrial morphology.

Aspartic acid supplementation ameliorates symptoms of diabetic kidney disease in mice.

Diabetic kidney disease (DKD) is among the most common and serious complications of both type 1 and type 2 diabetes. In this study, we used KK/Ta-Ins2Akita (KK-Akita) mice as a model of DKD and KK/Ta (KK) mice as controls to identify novel factors related to the development/progression of DKD. Capillary electrophoresis coupled with mass spectrometry analysis revealed that circulating Asp (l-aspartic acid) levels in diabetic KK-Akita mice tend to be lower than those in control KK mice. Therefore, we evaluated the effect of Asp supplementation to prevent the progression of DKD in KK-Akita mice. Mice were divided into three groups: (a) untreated KK mice (Control group), (b) untreated KK-Akita mice (DKD group), and (c) treated (double-volume Asp diet) KK-Akita mice (Tx group). Kidney sections were stained with fluorescein isothiocyanate-labeled lectins, wheat germ agglutinin (WGA), and anti-endothelial nitric oxide synthase (eNOS) antibody for evaluation of endothelial surface layer (ESL) and NO synthesis. The mesangial area and glomerular size in the DKD group were significantly larger than those in the Control group; however, there was no significant difference in those between the DKD and Tx groups. Albuminuria, the ratio of foot process effacement, and thickness of glomerular basement membrane in the Tx group were significantly lower than those in the DKD group. Furthermore, the expression levels of glomerular WGA and microvascular eNOS in the Tx group improved significantly and approached the level in the Control group. In conclusion, the improvement of albuminuria in the Tx group may be caused by the reduction of oxidative stress in the kidneys, which may lead to the subsequent improvement of glomerular ESL.

Circulating kidney injury molecule-1 as a biomarker of renal parameters in diabetic kidney disease.

AIMS/INTRODUCTION: Urinary kidney injury molecule-1 (KIM-1) has been associated with proximal tubular damage in human and animal studies. Although it has been recognized as a biomarker of acute kidney injury and chronic kidney disease, its significance in the serum remains unclear. Therefore, we examined the relationship of serum and urinary KIM-1 levels with renal parameters in patients with type 2 diabetes. MATERIALS AND METHODS: Serum and urinary KIM-1 levels, together with urinary liver-type fatty acid-binding protein, were measured in 602 patients with type 2 diabetes and an estimated glomerular filtration rate (eGFR) ≥30 mL/min/1.73 m2 . These were then compared with the urinary albumin-to-creatinine ratio and eGFR. RESULTS: The serum and urinary KIM-1 levels were significantly different among the three (eGFR ≥60, 45-59, <45 mL/min/1.73 m2 ) groups. These levels were positively associated with the albumin-to-creatinine ratio and negatively associated with eGFR. In a multivariate logistic model, both serum and urinary KIM-1 were associated with an increased albumin-to-creatinine ratio (>30 mg/g Cr), but only the serum KIM-1 was associated with a lower eGFR (<60 mL/min/1.73 m2 ), after adjustment for covariates. CONCLUSIONS: Renal parameters appear to be strongly associated with serum KIM-1, and not urinary KIM-1, in patients with type 2 diabetes and an eGFR ≥30 mL/min/1.73 m2 .

Asymmetric Synthesis of γ-Amino Alcohols by Copper-Catalyzed Hydroamination.

Asymmetric synthesis of γ-amino alcohols from unprotected allylic alcohols by a copper-catalyzed hydroamination strategy has been developed. Using easily accessible starting materials, a range of chiral 1,3-amino alcohols were prepared with excellent regio- and enantioselectivity. Further, this protocol provided an efficient one-step method for the enantioselective synthesis of γ-amino alcohols in an intermolecular manner.

Long-Term Intake of Glucoraphanin-Enriched Kale Suppresses Skin Aging via Activating Nrf2 and the TßRII/Smad Pathway in SAMP1 Mice.

Sulforaphane, a potent antioxidant compound, is unstable at ambient temperature, whereas its precursor glucoraphanin is stable and metabolized to sulforaphane. Thus, we hypothesized that glucoraphanin-rich diet could effectively induce antioxidant enzyme activities and investigated the protective effects of long-term intake of a glucoraphanin-enriched kale (GEK) diet on skin aging in senescence-accelerated mouse prone 1 (SAMP1) mice. The senescence grading score was significantly lower after treatment with GEK for 39 weeks than that of the control mice. GEK also suppressed the thinning of the dorsal skin layer. Moreover, the GEK treatment enhanced the collagen production and increased the nuclear translocation of Nrf2 and HO-1 expression level in the skin tissue. TßRII and Smad3 expressions were clearly higher in the GEK-treated group than in the control group. Thus, GEK suppressed senescence in SAMP1 mice by enhancing the antioxidant activity and collagen production via the TßRII/Smad3 pathway, suggesting its practical applications for protection against skin aging.

Regio- and Enantioselective Synthesis of 1,2-Diamine Derivatives by Copper-Catalyzed Hydroamination.

A highly regio- and enantioselective synthesis of 1,2-diamine derivatives from γ-substituted allylic pivalamides using copper-catalyzed hydroamination is reported. The N-pivaloyl group is essential, in both facilitating the hydrocupration step and suppressing an unproductive ß-elimination from the alkylcopper intermediate. This approach enables an efficient construction of chiral differentially protected vicinal diamines under mild conditions with broad functional group tolerance.

Long-Term Diet Supplementation with Lactobacillus paracasei K71 Prevents Age-Related Cognitive Decline in Senescence-Accelerated Mouse Prone 8.

This study aimed to assess the suppressive effect of long-term diet supplementation with Lactobacillus strains on cognitive decline in the senescence-accelerated mouse prone 8 (SAMP8) model. For 43 weeks, fourteen-week-old female SAMP8 mice were fed a standard diet containing 0.05% (w/w) Lactobacillus casei subsp. casei 327 (L. 327) or Lactobacillusparacasei K71 (L. K71) derived from rice grains and sake lees, respectively. SAMP8 mice that were fed a L. K71-supplemented diet had better cognitive performance compared with the control and L. 327 groups in the Barnes maze and passive avoidance tests. An ELISA analysis revealed that the levels of serotonin were elevated in the serum and brain tissue of L. K71-fed mice. The protein expression levels of brain-derived neurotrophic factor (BDNF), cAMP response element binding protein (CREB), and phosphorylated CREB were evaluated using western blot. Long-term administration of L. K71 resulted in increased protein expression of BDNF and CREB phosphorylation in the hippocampus. These results suggest that prolonged intake of a diet supplemented with a Lactobacillus strain derived from sake lees may prevent age-dependent cognitive decline by upregulating BDNF expression in the hippocampus.

A Modified System for the Synthesis of Enantioenriched N-Arylamines through Copper-Catalyzed Hydroamination.

Despite significant recent progress in copper-catalyzed enantioselective hydroamination chemistry, the synthesis of chiral N-arylamines, which are frequently found in natural products and pharmaceuticals, has not been realized. Initial experiments with N-arylhydroxylamine ester electrophiles were unsuccessful and, instead, their reduction in the presence of copper hydride (CuH) catalysts was observed. Herein, we report key modifications to our previously reported hydroamination methods that lead to broadly applicable conditions for the enantioselective net addition of secondary anilines across the double bond of styrenes, 1,1-disubstituted olefins, and terminal alkenes. NMR studies suggest that suppression of the undesired reduction pathway is the basis for the dramatic improvements in yield under the reported method.

Beneficial effects of tonsillectomy plus steroid pulse therapy on inflammatory and tubular markers in patients with IgA nephropathy.

BACKGROUND: IgA nephropathy (IgAN) is the most common form of primary glomerulonephritis worldwide. Tonsillectomy plus steroid pulse therapy has been able to induce clinical remission in early-stage IgAN. However, its possible effect on systemic and local cytokines and tubular markers has not been fully investigated. METHODS: We obtained serum and urine samples from 38 patients just before renal biopsy and third steroid pulse therapy. Markers of tubular damage such as N-acetyl-ß-d-glucosaminidase, and kidney injury molecule-1 and inflammation such as interleukin (IL)-6, monocyte chemotactic protein (MCP)-1, intercellular adhesion molecule (ICAM)-1, and vascular cell adhesion molecule (VCAM)-1 were measured by immunoassay. RESULTS: Before renal biopsy, only urinary inflammatory markers, except MCP-1, were associated with glomerular (proteinuria) and/or tubular damage markers. Proteinuria, hematuria, and estimated glomerular filtration rate dramatically improved after therapy. In addition, levels of serum IL-6 and ICAM-1 and all urinary markers declined significantly; however, serum MCP-1 and VCAM-1 levels did not. None of the urinary markers correlated with the serum inflammatory markers. CONCLUSION: Tonsillectomy plus steroid pulse therapy for patients with IgAN might be useful for improving not only glomerular damage marker but also tubular damage markers through the improvement of local renal inflammation.

Rapid and efficient copper-catalyzed Finkelstein reaction of (hetero)aromatics under continuous-flow conditions.

A general, rapid, and efficient method for the copper-catalyzed Finkelstein reaction of (hetero)aromatics has been developed using continuous flow to generate a variety of aryl iodides. The described method can tolerate a broad spectrum of functional groups, including N-H and O-H groups. Additionally, in lieu of isolation, the aryl iodide solutions were used in two distinct multistep continuous-flow processes (amidation and Mg-I exchange/nucleophilic addition) to demonstrate the flexibility of this method.

Iron-catalyzed directed alkylation of aromatic and olefinic carboxamides with primary and secondary alkyl tosylates, mesylates, and halides.

Alkenes, arenes, and heteroarenes possessing an 8-quinolylamide group as the directing group are alkylated with primary and secondary alkyl tosylates, mesylate, and halides in the presence of Fe(acac)3/diphosphine as a catalyst and ArZnBr as a base. The reaction proceeds stereospecifically for alkene substrates and takes place without loss of regiochemical integrity of the starting secondary tosylate, but with loss of the stereochemistry of the chiral center.

Fluorescent azadipyrrinato zinc(II) complex: hybridisation with a dipyrrinato ligand.

We synthesised heteroleptic azadipyrrinato-dipyrrinato hybrid zinc(II) complex 1-Zn-2, by means of the stepwise coordination method. Homoleptic bis(azadipyrrinato)zinc(II) complex 1-Zn-1 was non-fluorescent, whereas 1-Zn-2 exhibited detectable fluorescence from azadipyrrinato ligand 1.

Acerogenin A, a natural compound isolated from Acer nikoense Maxim, stimulates osteoblast differentiation through bone morphogenetic protein action.

We investigated the effects of acerogenin A, a natural compound isolated from Acer nikoense Maxim, on osteoblast differentiation by using osteoblastic cells. Acerogenin A stimulated the cell proliferation of MC3T3-E1 osteoblastic cells and RD-C6 osteoblastic cells (Runx2-deficient cell line). It also increased alkaline phosphatase activity in MC3T3-E1 and RD-C6 cells and calvarial osteoblastic cells isolated from the calvariae of newborn mice. Acerogenin A also increased the expression of mRNAs related to osteoblast differentiation, including Osteocalcin, Osterix and Runx2 in MC3T3-E1 cells and primary osteoblasts: it also stimulated Osteocalcin and Osterix mRNA expression in RD-C6 cells. The acerogenin A treatment for 3days increased Bmp-2, Bmp-4, and Bmp-7 mRNA expression levels in MC3T3-E1 cells. Adding noggin, a BMP specific-antagonist, inhibited the acerogenin A-induced increase in the Osteocalcin, Osterix and Runx2 mRNA expression levels. These results indicated that acerogenin A stimulates osteoblast differentiation through BMP action, which is mediated by Runx2-dependent and Runx2-independent pathways.

CCN3 and bone marrow cells.

CCN3 expression was observed in a broad variety of tissues from the early stage of development. However, a kind of loss of function in mice (CCN3 del VWC domain -/-) demonstrated mild abnormality, which indicates that CCN3 may not be critical for the normal embryogenesis as a single gene. The importance of CCN3 in bone marrow environment becomes to be recognized by the studies of hematopoietic stem cells and Chronic Myeloid Leukemia cells. CCN3 expression in bone marrow has been denied by several investigations, but we found CCN3 positive stromal and hematopoietic cells at bone extremities with a new antibody although they are a very few populations. We investigated the expression pattern of CCN3 in the cultured bone marrow derived mesenchymal stem cells and found its preference for osteogenic differentiation. From the analyses of in vitro experiment using an osteogenic mesenchymal stem cell line, Kusa-A1, we found that CCN3 downregulates osteogenesis by two different pathways; suppression of BMP and stimulation of Notch. Secreted CCN3 from Kusa cells inhibited the differentiation of osteoblasts in separate culture, which indicates the paracrine manner of CCN3 activity. CCN3 may also affect the extracellular environment of the niche for hematopoietic stem cells.