Towards understanding non-equivalence of α and β subunits within human hemoglobin in conformational relaxation and molecular oxygen rebinding

Picosecond to millisecond laser time-resolved transient absorption spectroscopy was used to study molecular oxygen (O₂) rebinding and conformational relaxation following O₂ photodissociation in the α and β subunits within human hemoglobin in the quaternary R-like structure. Oxy-cyanomet valency hybrids, α₂(Fe²⁺–O₂)β₂(Fe³⁺–CN) and α₂(Fe³⁺–CN)β₂(Fe²⁺–O₂), were used as models for oxygenated R-state hemoglobin. An extended kinetic model for geminate O₂ rebinding in the ferrous hemoglobin subunits, ligand migration between the primary and secondary docking site(s), and nonexponential tertiary relaxation within the R quaternary structure, was introduced and discussed. Significant functional non-equivalence of the α and β subunits in both the geminate O₂ rebinding and concomitant structural relaxation was revealed. For the β subunits, the rate constant for the geminate O₂ rebinding to the unrelaxed tertiary structure and the tertiary transition rate were found to be greater than the corresponding values for the α subunits. The conformational relaxation following the O₂ photodissociation in the α and β subunits was found to decrease the rate constant for the geminate O₂ rebinding, this effect being more than one order of magnitude greater for the β subunits than for the α subunits. Evidence was provided for the modulation of the O₂ rebinding to the individual α and β subunits within human hemoglobin in the R-state structure by the intrinsic heme reactivity through a change in proximal constraints upon the relaxation of the tertiary structure on a picosecond to microsecond time scale. Our results demonstrate that, for native R-state oxyhemoglobin, O₂ rebinding properties and spectral changes following the O₂ photodissociation can be adequately described as the sum of those for the α and β subunits within the valency hybrids. The isolated β chains (hemoglobin H) show similar behavior to the β subunits within the valency hybrids and can be used as a model for the β subunits within the R-state oxyhemoglobin. At the same time, the isolated α chains behave differently to the α subunits within the valency hybrids.

O₂ rebinding and conformational relaxation following O₂ photodissociation were studied on picosecond to millisecond time scale in the α and β subunits within human hemoglobin in the quaternary R-like structure.     

New 4-Aminoproline-Based Small Molecule Cyclopeptidomimetics as Potential Modulators of α4β1 Integrin

Integrin α₄β₁ belongs to the leukocyte integrin family and represents a therapeutic target of relevant interest given its primary role in mediating inflammation, autoimmune pathologies and cancer-related diseases. The focus of the present work is the design, synthesis and characterization of new peptidomimetic compounds that are potentially able to recognize α₄β₁ integrin and interfere with its function. To this aim, a collection of seven new cyclic peptidomimetics possessing both a 4-aminoproline (Amp) core scaffold grafted onto key α₄β₁-recognizing sequences and the (2-methylphenyl)ureido-phenylacetyl (MPUPA) appendage, was designed, with the support of molecular modeling studies. The new compounds were synthesized through SPPS procedures followed by in-solution cyclization maneuvers. The biological evaluation of the new cyclic ligands in cell adhesion assays on Jurkat cells revealed promising submicromolar agonist activity in one compound, namely, the c[Amp(MPUPA)Val-Asp-Leu] cyclopeptide. Further investigations will be necessary to complete the characterization of this class of compounds.     

Stereodivergent entry to β-branched β-trifluoromethyl α-amino acid derivatives by sequential catalytic asymmetric reactions

Currently, conventional reductive catalytic methodologies do not guarantee general access to enantioenriched β-branched β-trifluoromethyl α-amino acid derivatives. Herein, a one-pot approach to these important α-amino acids, grounded on the reduction – ring opening of Erlenmeyer–Plöchl azlactones, is presented. The configurations of the two chirality centers of the products are established during each of the two catalytic steps, enabling a stereodivergent process.

A one-pot approach to β-branched β-trifluoromethyl α-amino acids, grounded on the reduction – ring opening of Erlenmeyer–Plöchl azlactones, and complementary to conventional catalytic asymmetric hydrogenation, is presented.     

The Amyloid Fibril-Forming β-Sheet Regions of Amyloid β and α-Synuclein Preferentially Interact with the Molecular Chaperone 14-3-3ζ

14-3-3 proteins are abundant, intramolecular proteins that play a pivotal role in cellular signal transduction by interacting with phosphorylated ligands. In addition, they are molecular chaperones that prevent protein unfolding and aggregation under cellular stress conditions in a similar manner to the unrelated small heat-shock proteins. In vivo, amyloid β (Aβ) and α-synuclein (α-syn) form amyloid fibrils in Alzheimer’s and Parkinson’s diseases, respectively, a process that is intimately linked to the diseases’ progression. The 14-3-3ζ isoform potently inhibited in vitro fibril formation of the 40-amino acid form of Aβ (Aβ₄₀) but had little effect on α-syn aggregation. Solution-phase NMR spectroscopy of ¹⁵N-labeled Aβ₄₀ and A53T α-syn determined that unlabeled 14-3-3ζ interacted preferentially with hydrophobic regions of Aβ₄₀ (L11-H21 and G29-V40) and α-syn (V3-K10 and V40-K60). In both proteins, these regions adopt β-strands within the core of the amyloid fibrils prepared in vitro as well as those isolated from the inclusions of diseased individuals. The interaction with 14-3-3ζ is transient and occurs at the early stages of the fibrillar aggregation pathway to maintain the native, monomeric, and unfolded structure of Aβ₄₀ and α-syn. The N-terminal regions of α-syn interacting with 14-3-3ζ correspond with those that interact with other molecular chaperones as monitored by in-cell NMR spectroscopy.     

Synthesis of unstrained Criegee intermediates: inverse α-effect and other protective stereoelectronic forces can stop Baeyer–Villiger rearrangement of γ-hydroperoxy-γ-peroxylactones

How far can we push the limits in removing stereoelectronic protection from an unstable intermediate? We address this question by exploring the interplay between the primary and secondary stereoelectronic effects in the Baeyer–Villiger (BV) rearrangement by experimental and computational studies of γ-OR-substituted γ-peroxylactones, the previously elusive non-strained Criegee intermediates (CI). These new cyclic peroxides were synthesized by the peroxidation of γ-ketoesters followed by in situ cyclization using a BF₃·Et₂O/H₂O₂ system. Although the primary effect (alignment of the migrating C–R_m bond with the breaking O–O bond) is active in the 6-membered ring, weakening of the secondary effect (donation from the OR lone pair to the breaking C–R_m bond) provides sufficient kinetic stabilization to allow the formation and isolation of stable γ-hydroperoxy-γ-peroxylactones with a methyl-substituent in the C6-position. Furthermore, supplementary protection is also provided by reactant stabilization originating from two new stereoelectronic factors, both identified and quantified for the first time in the present work. First, an unexpected boat preference in the γ-hydroperoxy-γ-peroxylactones weakens the primary stereoelectronic effects and introduces a ∼2 kcal mol⁻¹ Curtin–Hammett penalty for reacquiring the more reactive chair conformation. Second, activation of the secondary stereoelectronic effect in the TS comes with a ∼2–3 kcal mol⁻¹ penalty for giving up the exo-anomeric stabilization in the 6-membered Criegee intermediate. Together, the three new stereoelectronic factors (inverse α-effect, misalignment of reacting bonds in the boat conformation, and the exo-anomeric effect) illustrate the richness of stereoelectronic patterns in peroxide chemistry and provide experimentally significant kinetic stabilization to this new class of bisperoxides. Furthermore, mild reduction of γ-hydroperoxy-γ-peroxylactone with Ph₃P produced an isolable γ-hydroxy-γ-peroxylactone, the first example of a structurally unencumbered CI where neither the primary nor the secondary stereoelectronic effect are impeded. Although this compound is relatively unstable, it does not undergo the BV reaction and instead follows a new mode of reactivity for the CI – a ring-opening process.

Protecting stereoelectronic effects prevent Baeyer–Villiger rearrangement and stabilize γ-OX-γ-peroxylactones (X = H, OH), the previously elusive non-strained Criegee intermediates.     

PET Diagnostic Molecules Utilizing Multimeric Cyclic RGD Peptide Analogs for Imaging Integrin αvβ3 Receptors

Multimeric ligands consisting of multiple pharmacophores connected to a single backbone have been widely investigated for diagnostic and therapeutic applications. In this review, we summarize recent developments regarding multimeric radioligands targeting integrin α_vβ₃ receptors on cancer cells for molecular imaging and diagnostic applications using positron emission tomography (PET). Integrin α_vβ₃ receptors are glycoproteins expressed on the cell surface, which have a significant role in tumor angiogenesis. They act as receptors for several extracellular matrix proteins exposing the tripeptide sequence arginine-glycine-aspartic (RGD). Cyclic RDG peptidic ligands c(RGD) have been developed for integrin α_vβ₃ tumor-targeting positron emission tomography (PET) diagnosis. Several c(RGD) pharmacophores, connected with the linker and conjugated to a chelator or precursor for radiolabeling with different PET radionuclides (¹⁸F, ⁶⁴Cu, and ⁶⁸Ga), have resulted in multimeric ligands superior to c(RGD) monomers. The binding avidity, pharmacodynamic, and PET imaging properties of these multimeric c(RGD) radioligands, in relation to their structural characteristics are analyzed and discussed. Furthermore, specific examples from preclinical studies and clinical investigations are included.     

Deoxygenative α-alkylation and α-arylation of 1,2-dicarbonyls

Construction of C–C bonds at the α-carbon is a challenging but synthetically indispensable approach to α-branched carbonyl motifs that are widely represented among drugs, natural products, and synthetic intermediates. Here, we describe a simple approach to generation of boron enolates in the absence of strong bases that allows for introduction of both α-alkyl and α-aryl groups in a reaction of readily accessible 1,2-dicarbonyls and organoboranes. Obviation of unselective, strongly basic and nucleophilic reagents permits carrying out the reaction in the presence of electrophiles that intercept the intermediate boron enolates, resulting in two new α-C–C bonds in a tricomponent process.

α-Branched carboxylic acids and other carbonyls are readily accessed by a metal- and base-free deoxygenative α-alkylation and α-arylation of 1,2-dicarbonyls via boron enolates, resulting in a tricomponent coupling with unconventional electrophiles.     

Alterations in β-Cell Sphingolipid Profile Associated with ER Stress and iPLA2β: Another Contributor to β-Cell Apoptosis in Type 1 Diabetes

Type 1 diabetes (T1D) development, in part, is due to ER stress-induced β-cell apoptosis. Activation of the Ca²⁺-independent phospholipase A₂ beta (iPLA₂β) leads to the generation of pro-inflammatory eicosanoids, which contribute to β-cell death and T1D. ER stress induces iPLA₂β-mediated generation of pro-apoptotic ceramides via neutral sphingomyelinase (NSMase). To gain a better understanding of the impact of iPLA₂β on sphingolipids (SLs), we characterized their profile in β-cells undergoing ER stress. ESI/MS/MS analyses followed by ANOVA/Student’s t-test were used to assess differences in sphingolipids molecular species in Vector (V) control and iPLA₂β-overexpressing (OE) INS-1 and Akita (AK, spontaneous model of ER stress) and WT-littermate (AK-WT) β-cells. As expected, iPLA₂β induction was greater in the OE and AK cells in comparison with V and WT cells. We report here that ER stress led to elevations in pro-apoptotic and decreases in pro-survival sphingolipids and that the inactivation of iPLA₂β restores the sphingolipid species toward those that promote cell survival. In view of our recent finding that the SL profile in macrophages—the initiators of autoimmune responses leading to T1D—is not significantly altered during T1D development, we posit that the iPLA₂β-mediated shift in the β-cell sphingolipid profile is an important contributor to β-cell death associated with T1D.     

Isoorientin Inhibits Amyloid β25–35-Induced Neuronal Inflammation in BV2 Cells by Blocking the NF-κB Signaling Pathway

Alzheimer’s disease (AD) is a severe neurodegenerative disorder. AD is pathologically characterized by the formation of intracellular neurofibrillary tangles, and extracellular amyloid plaques which were comprised of amyloid-beta (Aβ) peptides. Aβ induces neurodegeneration by activating microglia, which triggers neurotoxicity by releasing various inflammatory mediators and reactive oxygen species (ROS). Nuclear factor-kappa B (NF-κB) is expressed in human tissues including the brain and plays an important role in Aβ-mediated neuronal inflammation. Thus, the identification of molecules that inhibit the NF-κB pathway is considered an attractive strategy for the treatment and prevention of AD. Isoorientin (3′,4′,5,7-Tetrahydroxy-6-C-glucopyranosyl flavone; ISO), which can be extracted from several plant species, such as Philostachys and Patrinia is known to have various pharmacological activities such as anticancer, antioxidant, and antibacterial activity. However, the effect of ISO on Aβ-mediated inflammation and apoptosis in the brain has yet to be elucidated. In the present study, we investigated whether ISO regulated Aβ-induced neuroinflammation in microglial cells and further explored the underlying mechanisms. Our results showed that ISO inhibited the expression of iNOS and COX-2 induced by Aβ_25–35. And, it inhibited the secretion of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). In addition, ISO reduced the ROS production in Aβ_25–35-induced BV2 cells and inhibited NF-κB activation. Furthermore, ISO blocked Aβ_25–35-induced apoptosis of BV2 cells. Based on these findings, we suggest that ISO represents a promising therapeutic drug candidate for the treatment and prevention of AD.     

Cyclic RGD and isoDGR Integrin Ligands Containing cis-2-amino-1-cyclopentanecarboxylic (cis-β-ACPC) Scaffolds

Integrin ligands containing the tripeptide sequences Arg-Gly-Asp (RGD) and iso-Asp-Gly- Arg (isoDGR) were actively investigated as inhibitors of tumor angiogenesis and directing unit in tumor-targeting drug conjugates. Reported herein is the synthesis, of two RGD and one isoDGR cyclic peptidomimetics containing (1S,2R) and (1R,2S) cis-2-amino-1-cyclopentanecarboxylic acid (cis-β-ACPC), using a mixed solid phase/solution phase synthetic protocol. The three ligands were examined in vitro in competitive binding assays to the purified α_vβ₃ and α₅β₁ receptors using biotinylated vitronectin (α_vβ₃) and fibronectin (α₅β₁) as natural displaced ligands. The IC50 values of the ligands ranged from nanomolar (the two RGD ligands) to micromolar (the isoDGR ligand) with a pronounced selectivity for α_vβ₃ over α₅β₁. In vitro cell adhesion assays were also performed using the human skin melanoma cell line WM115 (rich in integrin α_vβ₃). The two RGD ligands showed IC₅₀ values in the same micromolar range as the reference compound (cyclo[RGDfV]), while for the isoDGR derivative an IC₅₀ value could not be measured for the cell adhesion assay. A conformational analysis of the free RGD and isoDGR ligands by NMR (VT-NMR and NOESY experiments) and computational studies (MC/EM and MD), followed by docking simulations performed in the α_Vβ₃ integrin active site, provided a rationale for the behavior of these ligands toward the receptor.     

Pharmacokinetic Characterization of LW6, a Novel Hypoxia-Inducible Factor-1α (HIF-1α) Inhibitor in Mice

LW6, an (aryloxyacetylamino)benzoic acid derivative, was recently identified to be an inhibitor of hypoxia-inducible factor-1α (HIF-1α), which is an attractive target for cancer therapeutics. Although LW6 is known to act by inhibiting the accumulation of HIF-1α, pharmacokinetics needs to be evaluated to assess its potential as an anti-tumor agent. Here, we investigated the plasma pharmacokinetics and metabolism of LW6 in mice. LW6 exhibited a small volume of distribution (0.5 ± 0.1 L/kg), and a short terminal half-life (0.6 ± 0.1 h). Following intravenous or oral administration, LW6 was rapidly converted to its active metabolite, (4-adamantan-1-yl-phenoxy)acetic acid (APA). Although LW6 was rapidly absorbed, its oral bioavailability, estimated using AUC_last values, was low (1.7 ± 1.8%). It was slowly degraded in mouse liver microsomes (t_1/2 > 1 h) and serum (t_1/2 > 6 h). About 54% or 44.8% of LW6 was available systemically as APA in the mouse after a single intravenous or oral administration, respectively. Thus, our results indicated the need to simultaneously consider the active metabolite as well as the parent compound for successful evaluation during lead optimization.     

Four Novel Phenanthrene Derivatives with α-Glucosidase Inhibitory Activity from Gastrochilus bellinus

Four new phenanthrene derivatives, gastrobellinols A-D (1–4), were isolated from the methanolic extract of Gastrochilus bellinus (Rchb.f.) Kuntze, along with eleven known phenolic compounds including agrostophyllin (5), agrostophyllidin (6), coniferyl aldehyde (7), 4-hydroxybenzaldehyde (8), agrostophyllone (9), gigantol (10), 4-(methoxylmethyl)phenol (11), syringaldehyde (12), 1-(4′-hydroxybenzyl)-imbricartin (13), 6-methoxycoelonin (14), and imbricatin (15). Their structures were determined by spectroscopic methods. Each isolate was evaluated for α-glucosidase inhibitory activity. Compounds 1, 2, 3, 7, 9, 13, and 15 showed higher activity than the drug acarbose. Gastrobellinol C (3) exhibited the strongest α-glucosidase inhibition with an IC₅₀ value of 45.92 μM. A kinetic study of 3 showed competitive inhibition on the α-glucosidase enzyme. This is the first report on the phytochemical constituents and α-glucosidase inhibitory activity of G. bellinus.     

α-Amyrin and β-Amyrin Isolated from Celastrus hindsii Leaves and Their Antioxidant,Anti-Xanthine Oxidase,and Anti-Tyrosinase Potentials

Celastrus hindsii is a popular medicinal plant in Vietnam and Southeast Asian countries as well as in South America. In this study, an amount of 12.05 g of an α-amyrin and β-amyrin mixture was isolated from C. hindsii (10.75 g/kg dry weight) by column chromatography applying different solvent systems to obtain maximum efficiency. α-Amyrin and β-amyrin were then confirmed by gas chromatography-mass spectrometry (GC-MS), electrospray ionization-mass spectrometry (ESI-MS), and nuclear magnetic resonance (NMR). The antioxidant activities of the α-amyrin and β-amyrin mixture were determined via 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,20-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays with IC₅₀ of 125.55 and 155.28 µg/mL, respectively. The mixture exhibited a high potential for preventing gout by inhibiting a relevant key enzyme, xanthine oxidase (XO) (IC₅₀ = 258.22 µg/mL). Additionally, an important enzyme in skin hyperpigmentation, tyrosinase, was suppressed by the α-amyrin and β-amyrin mixture (IC₅₀ = 178.85 µg/mL). This study showed that C. hindsii is an abundant source for the isolation of α-amyrin and β-amyrin. Furthermore, this was the first study indicating that α-amyrin and β-amyrin mixture are promising in future therapies for gout and skin hyperpigmentation.     

Redox deracemization of β,γ-alkynyl α-amino esters

The first non-enzymatic redox deracemization method using molecular oxygen as the terminal oxidant has been described. The one-pot deracemization of β,γ-alkynyl α-amino esters consisted of a copper-catalyzed aerobic oxidation and chiral phosphoric acid-catalyzed asymmetric transfer hydrogenation with excellent functional group compatibility. By using benzothiazoline as the reducing reagent, an exclusive chemoselectivity at the C N bond over the C C bond was achieved, allowing for efficient deracemization of a series of α-amino esters bearing diverse α-alkynyl substituent patterns. The origins of chemo- and enantio-selectivities were elucidated by experimental and computational mechanistic investigation. The generality of the strategy is further demonstrated by efficient deracemization of β,γ-alkenyl α-amino esters.

A one-pot deracemization of β,γ-alkynyl α-amino esters consisting of an aerobic oxidation and chiral phosphoric acid-catalyzed asymmetric transfer hydrogenation has been described.     

Discovery of Amide-Functionalized Benzimidazolium Salts as Potent α-Glucosidase Inhibitors

α-Glucosidase inhibitors (AGIs) are used as medicines for the treatment of diabetes mellitus. The α-Glucosidase enzyme is present in the small intestine and is responsible for the breakdown of carbohydrates into sugars. The process results in an increase in blood sugar levels. AGIs slow down the digestion of carbohydrates that is helpful in controlling the sugar levels in the blood after meals. Among heterocyclic compounds, benzimidazole moiety is recognized as a potent bioactive scaffold for its wide range of biologically active derivatives. The aim of this study is to explore the α-glucosidase inhibition ability of benzimidazolium salts. In this study, two novel series of benzimidazolium salts, i.e., 1-benzyl-3-{2-(substituted) amino-2-oxoethyl}-1H-benzo[d]imidazol-3-ium bromide 9a–m and 1-benzyl-3-{2-substituted) amino-2-oxoethyl}-2-methyl-1H-benzo[d] imidazol-3-ium bromide 10a–m were screened for their in vitro α-glucosidase inhibitory potential. These compounds were synthesized through a multistep procedure and were characterized by ¹H-NMR, ¹³C-NMR, and EI-MS techniques. Compound 10d was identified as the potent α-glucosidase inhibitor among the series with an IC₅₀ value of 14 ± 0.013 μM, which is 4-fold higher than the standard drug, acarbose. In addition, compounds 10a, 10e, 10h, 10g, 10k, 10l, and 10m also exhibited pronounced potential for α-glucosidase inhibition with IC₅₀ value ranging from 15 ± 0.037 to 32.27 ± 0.050 µM when compared with the reference drug acarbose (IC₅₀ = 58.8 ± 0.12 μM). A molecular docking study was performed to rationalize the binding interactions of potent inhibitors with the active site of the α-glucosidase enzyme.     

Preparation of α-amino acids via Ni-catalyzed reductive vinylation and arylation of α-pivaloyloxy glycine

This work emphasizes easy access to α-vinyl and aryl amino acids via Ni-catalyzed cross-electrophile coupling of bench-stable N-carbonyl-protected α-pivaloyloxy glycine with vinyl/aryl halides and triflates. The protocol permits the synthesis of α-amino acids bearing hindered branched vinyl groups, which remains a challenge using the current methods. On the basis of experimental and DFT studies, simultaneous addition of glycine α-carbon (Gly) radicals to Ni(0) and Ar–Ni(ii) may occur, with the former being more favored where oxidative addition of a C(sp²) electrophile to the resultant Gly–Ni(i) intermediate gives a key Gly–Ni(iii)–Ar intermediate. The auxiliary chelation of the N-carbonyl oxygen to the Ni center appears to be crucial to stabilize the Gly–Ni(i) intermediate.

We have developed Ni-catalyzed reductive coupling of N-carbonyl protected α-pivaloyloxy glycine with Csp²-electrophiles that enabled facile preparation of α-amino acids, including those bearing hindered branched vinyl groups.     

Square-Planar Heteroleptic Complexes of α-Diimine-NiII-Catecholate Type: Intramolecular Ligand-to-Ligand Charge Transfer

Two heteroleptic Ni^II complexes combined the redox-active catecholate and 2,2′- bipyridine ligand platforms were synthesized to observe a photoinduced intramolecular ligand-to-ligand charge transfer (LL’CT, HOMO_catecholate → LUMO_α-diimine). A molecular design of compound [Ni^II(3,6-Cat)(bipy)]∙CH₃CN (1) on the base of bulky 3,6-di-tert-butyl-o-benzoquinone (3,6-DTBQ) was an annelation of the ligand with an electron donor glycol fragment, producing derivative [Ni^II(3,6-Cat^gly)(bipy)]∙CH₂Cl₂ (2), in order to influence the energy of LL’CT transition. A substantial longwave shift of the absorption peak was observed in the UV-Vis-NIR spectra of 2 compared with those in 1. In addition, the studied Ni^II derivatives demonstrated a pronounced negative solvatochromism, which was established using a broad set of solvents. The molecular geometry of both compounds can be ascribed as an insignificantly distorted square-planar type, and the π–π intermolecular stacking of the neighboring α-diimines is realized in a crystal packing. There is a lamellar crystal structure for complex 1, whereas the perpendicular T-motifs with the inter-stacks attractive π–π interactions form the packing of complex 2. The redox-active nature of ligand systems was clearly shown through the electrochemical study: a quasi-reversible one-electron reduction of 2,2′-bipyridine and two reversible successive one-electron oxidative conversations (“catecholate dianion—o-benzosemiquinonato radical anion—neutral o-benzoquinone”) were detected.     

Cereblon contributes to the development of pulmonary fibrosis via inactivation of adenosine monophosphate-activated protein kinase α1

Pulmonary fibrosis is a progressive and lethal lung disease characterized by the proliferation and differentiation of lung fibroblasts and the accumulation of extracellular matrices. Since pulmonary fibrosis was reported to be associated with adenosine monophosphate-activated protein kinase (AMPK) activation, which is negatively regulated by cereblon (CRBN), we aimed to determine whether CRBN is involved in the development of pulmonary fibrosis. Therefore, we evaluated the role of CRBN in bleomycin (BLM)-induced pulmonary fibrosis in mice and in transforming growth factor-beta 1 (TGF-β1)-induced differentiation of human lung fibroblasts. BLM-induced fibrosis and the mRNA expression of collagen and fibronectin were increased in the lung tissues of wild-type (WT) mice; however, they were significantly suppressed in Crbn knockout (KO) mice. While the concentrations of TGF-β1/2 in bronchoalveolar lavage fluid were increased via BLM treatment, they were similar between BLM-treated WT and Crbn KO mice. Knockdown of CRBN suppressed TGF-β1-induced activation of small mothers against decapentaplegic 3 (SMAD3), and overexpression of CRBN increased it. TGF-β1-induced activation of SMAD3 increased α-smooth muscle actin (α-SMA) and collagen levels. CRBN was found to be colocalized with AMPKα1 in lung fibroblasts. CRBN overexpression inactivated AMPKα1. When cells were treated with metformin (an AMPK activator), the CRBN-induced activation of SMAD3 and upregulation of α-SMA and collagen expression were significantly suppressed, suggesting that increased TGF-β1-induced activation of SMAD3 via CRBN overexpression is associated with AMPKα1 inactivation. Taken together, these data suggest that CRBN is a profibrotic regulator and maybe a potential target for treating lung fibrosis.Subject terms: Pathogenesis, Biochemistry