Novel 1,4-Dihydropyridine Derivatives as Mineralocorticoid Receptor Antagonists.

The mineralocorticoid receptor (MR) belongs to the steroid receptor subfamily of nuclear receptors. MR is a transcription factor key in regulating blood pressure and mineral homeostasis. In addition, it plays an important role in a broad range of biological and pathological conditions, greatly expanding its interest as a pharmacological target. Non-steroidal MR antagonists (MRAs) are of particular interest to avoid side effects and achieve tissue-specific modulation of the receptor. The 1,4-dihydropyridine (1,4-DHP) ring has been identified as an appropriate scaffold to develop non-steroidal MRAs. We report the identification of a novel series of 1,4-DHP that has been guided by structure-based drug design, focusing on the less explored DHP position 2. Interestingly, substituents at this position might interfere with MR helix H12 disposition, which is essential for the recruitment of co-regulators. Several of the newly synthesized 1,4-DHPs show interesting properties as MRAs and have a good selectivity profile. These 1,4-DHPs promote MR nuclear translocation with less efficiency than the natural agonist aldosterone, which explains, at least in part, its antagonist character. Molecular dynamic studies are suggestive of several derivatives interfering with the disposition of H12 in the agonist-associated conformation, and thus, they might stabilize an MR conformation unable to recruit co-activators.

Synthesis and biological activity of cyclopropyl Δ7-dafachronic acids as DAF-12 receptor ligands.

The four cyclopropyl stereoisomers of Δ7-dafachronic acids were prepared from the bile acid hyodeoxycholic acid and employed as chemical tools to exploit the importance of the orientation and spatial disposition of the carboxyl tail and the C25-methyl group for the binding at the DAF-12 receptor. The synthesis route was based on (a) Walden inversion and stereoselective PtO2-hydrogenation to convert the L-shaped 5ß-cholanoid scaffold into the planar 5α-sterol intermediate; (b) two-carbon homologation of the side chain by Wittig and cyclopropanation reaction; and (c) formation of the 3-keto group and Δ7 double bond. The synthesized isomers were isolated and tested for their activity as DAF-12 ligands by AlphaScreen assays. Results showed a significant loss of potency and efficacy for all the four stereoisomers when compared to the parent endogenous ligand. Computational analysis has evidenced the configurational and conformational arrangement of both the carboxylic and the C25-methyl group of dafachronic acids as key structural determinants for DAF-12 binding and activation.

Investigating the allosteric reverse signalling of PARP inhibitors with microsecond molecular dynamic simulations and fluorescence anisotropy.

The inhibition of the poly(ADP-ribose) polymerase (PARP) family members is a strategy pursued for the development of novel therapeutic agents in a range of diseases, including stroke, cardiac ischemia, cancer, inflammation and diabetes. Even though some PARP-1 inhibitors have advanced to clinical setting for cancer therapy, a great deal of attention is being devoted to understand the polypharmacology of current PARP inhibitors. Besides blocking the catalytic activity, recent works have shown that some PARP inhibitors exhibit a poisoning activity, by trapping the enzyme at damaged sites of DNA and forming cytotoxic complexes. In this study we have used microsecond molecular dynamics to study the allosteric reverse signalling that is at the basis of such an effect. We show that Olaparib, but not Veliparib and HYDAMTIQ, is able to induce a specific conformational drift of the WGR domain of PARP-1, which stabilizes PARP-1/DNA complex through the locking of several salt bridge interactions. Fluorescence anisotropy assays support such a mechanism, providing the first experimental evidence that HYDAMTIQ, a potent PARP inhibitor with neuroprotective properties, is less potent than Olaparib to trap PARP-1/DNA complex.

Brain natriuretic peptide modulates calcium homeostasis and epidermal growth factor receptor gene signalling in asthmatic airways smooth muscle cells.

The airway epithelium acts as a barrier and provides a critical interface between the body and the external environment. Brain natriuretic peptide (BNP) plays an important role in several bronchial functions, including relaxation. BNP relaxes airways by binding and activating natriuretic peptide receptor-A expressed from the airway epithelium. Although relaxation effect has been extensively investigated, less is known about BNP-regulated intracellular biomolecular pathways leading to bronchial relaxation. To this aim, we investigated BNP effects on gene signalling of airway smooth muscle cells (ASM) obtained from donors with asthma by using a RT(2) profiler™ PCR array. When compared with control, treatment for 2 h with supernatant from BNP-treated (1 µM) bronchial epithelial cells (BEAS-2B) induced in asthmatic ASM cells a rapid reduction of transcription of EGFR and genes involving in actin and calcium homeostasis, as those of Protein kinase C (PKC) and RhoA-ROCK gene pathways. Immunofluorescence and western blotting did not shown any difference comparing control and ASM cells treated with conditioned medium from BNP-treated BEAS-2B. This study provides evidence that the effect of BNP on relaxing bronchial in ASM cells is mediated from epithelium and associates to rapid changes of EGFR and calcium homeostasis-associated gene levels.

Brain natriuretic peptide protects against hyperresponsiveness of human asthmatic airway smooth muscle via an epithelial cell-dependent mechanism.

Brain natriuretic peptide (BNP) relaxes airways by activating natriuretic peptide receptor-A and elevating cyclic guanosine monophosphate. BNP is more effective in passively sensitized human bronchi compared with control airways. The molecular and cellular patterns involved in this signaling are unknown. The aim of this study was to investigate the influence of BNP on airway smooth muscle (ASM) cells obtained from donors with asthma and healthy donors and to identify the mechanisms involved in BNP-mediated relaxation. The contractile response of ASM cells was microscopically assessed in vitro in the presence of 1 µM BNP or with supernatant from human bronchial epithelial (BEAS-2B) cells pretreated with 1 µM BNP. We investigated the role of muscarinic M2 receptors and inducible nitric oxide synthase (iNOS), quantified the release of acetylcholine and nitric oxide (NO), and assessed the gene/protein expression of iNOS and myosin phosphatase target subunit 1 (MYPT1). Supernatant from BEAS-2B cells treated with BNP reduced the hyperreactivity of asthmatic ASM cells by shifting the potency of histamine by 1.19-fold but had no effect in healthy ASM cells. BNP was not effective directly on ASM cells. Blocking muscarinic M2-receptors and iNOS abolished the protective role of supernatant from BEAS-2B treated with BNP. BNP stimulated the release of acetylcholine (210.7 ± 11.1%) from BEAS-2B cells that in turn increased MYPT1 and iNOS gene/protein expression and enhanced NO levels in asthmatic ASM supernatant (35.0 ± 13.0%). This study provides evidence that BNP protects against bronchial hyperresponsiveness via an interaction between respiratory epithelium and ASM in subjects with asthma.

Semisynthetic bile acid FXR and TGR5 agonists: physicochemical properties, pharmacokinetics, and metabolism in the rat.

We report on the relationship between the structure-pharmacokinetics, metabolism, and therapeutic activity of semisynthetic bile acid analogs, including 6α-ethyl-3α,7α-dihydroxy-5ß-cholan-24-oic acid (a selective farnesoid X receptor [FXR] receptor agonist), 6α-ethyl-23(S)-methyl-3α,7α,12α-trihydroxy-5ß-cholan-24-oic acid (a specific Takeda G protein-coupled receptor 5 [TGR5] receptor agonist), and 6α-ethyl-3α,7α-dihydroxy-24-nor-5ß-cholan-23-sulfate (a dual FXR/TGR5 agonist). We measured the main physicochemical properties of these molecules, including ionization constants, water solubility, lipophilicity, detergency, and protein binding. Biliary secretion and metabolism and plasma and hepatic concentrations were evaluated by high-pressure liquid chromatography-electrospray-mass spectrometry/mass spectrometry in bile fistula rat and compared with natural analogs chenodeoxycholic, cholic acid, and taurochenodexycholic acid and intestinal bacteria metabolism was evaluated in terms of 7α-dehydroxylase substrate-specificity in anaerobic human stool culture. The semisynthetic derivatives detergency, measured in terms of their critical micellar concentration, was quite similar to the natural analogs. They were slightly more lipophilic than the corresponding natural analogs, evaluated by their 1-octanol water partition coefficient (log P), because of the ethyl group in 6 position, which makes these molecules very stable toward bacterial 7-dehydroxylation. The hepatic metabolism and biliary secretion were different: 6α-ethyl-3α,7α-dihydroxy-5ß-cholan-24-oic acid, as chenodeoxycholic acid, was efficiently conjugated with taurine in the liver and, only in this form, promptly and efficiently secreted in bile. 6α-Ethyl-23(S)-methyl-3α,7α,12α-trihydroxy-5ß-cholan-24-oic acid was poorly conjugated with taurine because of the steric hindrance of the methyl at C23(S) position metabolized to the C23(R) isomer and partly conjugated with taurine. Conversely, 6α-ethyl-3α,7α-dihydroxy-24-nor-5ß-cholan-23-sulfate was secreted in bile unmodified and as 3-glucuronide. Therefore, minor structural modifications profoundly influence the metabolism and biodistribution in the target organs where these analogs exert therapeutic effects by interacting with FXR and/or TGR5 receptors.

Discovery and Structure-Activity Relationships of Novel ssDAF-12 Receptor Modulators.

The nuclear receptor ssDAF-12 has been recognized as the key molecular player regulating the life cycle of the nematode parasite Strongyloides stercoralis. ssDAF-12 ligands permit the receptor to function as an on/off switch modulating infection, making it vulnerable to therapeutic intervention. In this study, we report the design and synthesis of a set of novel dafachronic acid derivatives, which were used to outline the first structure-activity relationship targeting the ssDAF-12 receptor and to unveil hidden properties shared by the molecular shape of steroidal ligands that are relevant to the receptor binding and modulation. Moreover, biological results led to the discovery of sulfonamide 3 as a submicromolar ssDAF-12 agonist endowed with a high receptor selectivity, no toxicity, and improved properties, as well as to the identification of unprecedented ssDAF-12 antagonists that can be exploited in the search for novel chemical tools and alternative therapeutic approaches for treating parasitism such as Strongyloidiasis.

BH4 domain of bcl-2 protein is required for its proangiogenic function under hypoxic condition.

Beyond its classical role as apoptosis inhibitor, bcl-2 protein promotes tumor angiogenesis and the removal of N-terminal bcl-2 homology (BH4) domain abrogates bcl-2-induced hypoxia-inducible factor 1 (HIF-1)-mediated vascular endothelial growth factor (VEGF) expression in hypoxic cancer cells. Using M14 human melanoma cell line and its derivative clones stably overexpressing bcl-2 wild-type or deleted of its BH4 domain, we found that conditioned media (CM) from cells expressing BH4-deleted bcl-2 protein showed a reduced capability to increase in vitro human endothelial cells proliferation and differentiation, and in vivo neovascularization compared with CM from cells overexpressing wild-type bcl-2. Moreover, xenografts derived from cells expressing bcl-2 lacking BH4 domain showed a reduction of metastatic potential compared with tumors derived from wild-type bcl-2 transfectants injection. Stably expressing the Flag-tagged N-terminal sequence of bcl-2 protein, encompassing BH4 domain, we found that this domain is sufficient to enhance the proangiogenic HIF-1/VEGF axis under hypoxic condition. Indeed, lacking of BH4 domain abolishes the interaction between bcl-2 and HIF-1α proteins and the capability of exogenous bcl-2 protein to localize in the nucleus. Moreover, when endoplasmic reticulum-targeted bcl-2 protein is overexpressed in cells, this protein lost the capability to synergize with hypoxia to induce the proangiogenic HIF-1/VEGF axis as shown by wild-type bcl-2 protein. These results demonstrate that BH4 domain of bcl-2 is required for the ability of this protein to increase tumor angiogenesis and progression and indicate that bcl-2 nuclear localization may be required for bcl-2-mediated induction of HIF-1/VEGF axis.

Asymmetric synthesis of the four diastereoisomers of a novel non-steroidal farnesoid X receptor (FXR) agonist: role of the chirality on the biological activity.

An asymmetric synthetic strategy was designed for the preparation of the four possible diastereoisomers of 3,6-dimethyl-1-(2-methylphenyl)-4-(4-phenoxyphenyl)-4,8-dihydro-1H-pyrazolo[3,4-e][1,4]thiazepin-7-one, a non-steroidal FXR agonist, we recently discovered following a virtual screening approach. The results obtained from an AlphaScreen assay clearly demonstrated that only the isomer endowed with 4R,6S absolute configuration is responsible for the biological activity. A deep investigation of the different putative binding modes adopted by these enantiomerically pure ligands using computational modeling studies confirmed the enantioselectivity of FXR towards this class of molecules.

Chemical exploration of TGR5 functional hot-spots: Synthesis and structure-activity relationships of C7- and C23-Substituted cholic acid derivatives.

The activation of TGR5 bestows on bile acids the ability to modulate nongenomic signaling pathways, which are responsible of physiological actions including immunosuppressive and anti-inflammatory properties as well as the regulation of glucose metabolism and energy homeostasis. TGR5 agonists have therefore emerged in drug discovery and preclinical appraisals as promising compounds for the treatment of liver diseases and metabolic syndrome. In this study, we have been devising site-selected chemical modifications of the bile acid scaffold to provide novel chemical tools able to modulate the functions of TGR5 in different tissues. Biological results of the tested collection of semisynthetic cholic acid derivatives were used to extend the structure-activity relationships of TGR5 agonists and to clarify the molecular basis and functional role of TGR5 hot-spots in the receptor activation and selectivity. Some unexpected properties deriving from the molecular structure of bile acids have been unveiled as relevant to the receptor activation and may hence be used to design novel, selective and potent TGR5 agonists.

Novel pyridine-containing histone deacetylase inhibitors strongly arrest proliferation, induce apoptosis and modulate miRNAs in cancer cells.

After over 30 years of research, the development of HDAC inhibitors led to five FDA/Chinese FDA-approved drugs and many others under clinical or preclinical investigation to treat cancer and non-cancer diseases. Herein, based on our recent development of pyridine-based isomers as HDAC inhibitors, we report a series of novel 5-acylamino-2-pyridylacrylic- and -picolinic hydroxamates and 2'-aminoanilides 5-8 as anticancer agents. The hydroxamate 5d proved to be quite HDAC3/6-selective exhibiting IC50 values of 80 and 11 nM, respectively, whereas the congener 5e behaved as inhibitor of HDAC1-3, -6, -8, and -10 (class I/IIb-selective inhibitor) at nanomolar level. Compound 5e provided a huge antiproliferative activity (nanomolar IC50 values) against both haematological and solid cancer cell lines. In leukaemia U937 cells, the hydroxamate 5d and the 2'-aminoanilide 8f induced remarkable cell death after 48 h, with 76% and 100% pre-G1 phase arrest, respectively, showing a stronger effect with respect to SAHA and MS-275 used as reference compounds. In U937 cells, the highest dose- and time-dependent cytodifferentiation was obtained by the 2'-aminoanilide 8d (up to 35% of CD11c positive/propidium iodide negative cells at 5 µM for 48 h). The same 8d and the hydroxamates 5d and 5e were the most effective in inducing p21 protein expression in the same cell line. Mechanistically, 5d, 5e, 8d and 8f increased mRNA expression of p21, BAX and BAK, downregulated cyclin D1 and BCL-2 and modulated pro- and anti-apoptotic microRNAs towards apoptosis induction. Finally, 5e strongly arrested proliferation in nine different haematological cancer cell lines, with dual-digit nanomolar potency towards MV4-11, Kasumi-1, and NB4, being more potent than mocetinostat, used as reference drug.

Pyrazole[3,4-e][1,4]thiazepin-7-one derivatives as a novel class of Farnesoid X Receptor (FXR) agonists.

A virtual screening procedure was applied to the discovery of structurally diverse non-steroidal Farnesoid X Receptor (FXR) agonists. From 117 compounds selected by virtual screening, a total of 47 compounds were found to be FXR agonists, with 34 of them showing activity below a concentration of 20 µM. 1H-Pyrazole[3,4-e][1,4]thiazepin-7-one-based hit compound 7 was chosen for hit-to-lead optimization. A large number of 1H-pyrazole[3,4-e][1,4]thiazepin-7-one derivatives was designed, synthesized, and evaluated by a cell-based luciferase transactivation assay for their agonistic activity against FXR. Most of them exhibited low micromolar range of potency and very high efficacy.

Development of 3α,7α-dihydroxy-6α-ethyl-24-nor-5ß-cholan-23-sulfate sodium salt (INT-767): Process optimization, synthesis and characterization of metabolites.

Herein we report our synthetic efforts in supporting the development of the bile alcohol sulfate INT-767, a FXR/TGR5 dual agonist with remarkable therapeutic potential for liver disorders. We describe the process development to a final route for large scale preparation and analogues synthesis. Key sequences include Grignard addition, a one-pot two-step shortening-reduction of the carboxylic side chain, and the final sulfation reaction. The necessity for additional steps such as the protection/deprotection of hydroxyl groups at the steroidal body was also evaluated for step-economy and formation of side-products. Critical bottlenecks such as the side chain degradation have been tackled using flow technology before scaling-up individual steps. The final synthetic route may be successfully employed to produce the amount of INT-767 required to support late-stage clinical development of the compound. Furthermore, potential metabolites have been synthesized, characterized and evaluated for their ability to modulate FXR and TGR5 receptors providing key reference standards for future drug investigations, as well as offering further insights into the structure-activity relationships of this class of compounds.

First-in-Class Inhibitors of the Ribosomal Oxygenase MINA53.

MINA53 is a JmjC domain 2-oxoglutarate-dependent oxygenase that catalyzes ribosomal hydroxylation and is a target of the oncogenic transcription factor c-MYC. Despite its anticancer target potential, no small-molecule MINA53 inhibitors are reported. Using ribosomal substrate fragments, we developed mass spectrometry assays for MINA53 and the related oxygenase NO66. These assays enabled the identification of 2-(aryl)alkylthio-3,4-dihydro-4-oxoypyrimidine-5-carboxylic acids as potent MINA53 inhibitors, with selectivity over NO66 and other JmjC oxygenases. Crystallographic studies with the JmjC demethylase KDM5B revealed active site binding but without direct metal chelation; however, molecular modeling investigations indicated that the inhibitors bind to MINA53 by directly interacting with the iron cofactor. The MINA53 inhibitors manifest evidence for target engagement and selectivity for MINA53 over KDM4-6. The MINA53 inhibitors show antiproliferative activity with solid cancer lines and sensitize cancer cells to conventional chemotherapy, suggesting that further work investigating their potential in combination therapies is warranted.

Functional characterization of the semisynthetic bile acid derivative INT-767, a dual farnesoid X receptor and TGR5 agonist.

Two dedicated receptors for bile acids (BAs) have been identified, the nuclear hormone receptor farnesoid X receptor (FXR) and the G protein-coupled receptor TGR5, which represent attractive targets for the treatment of metabolic and chronic liver diseases. Previous work characterized 6α-ethyl-3α,7α-dihydroxy-5ß-cholan-24-oic acid (INT-747), a potent and selective FXR agonist, as well as 6α-ethyl-23(S)-methyl-3α,7α,12α-trihydroxy-5ß-cholan-24-oic acid (INT-777), a potent and selective TGR5 agonist. Here we characterize 6α-ethyl-3α,7α,23-trihydroxy-24-nor-5ß-cholan-23-sulfate sodium salt (INT-767), a novel semisynthetic 23-sulfate derivative of INT-747. INT-767 is a potent agonist for both FXR (mean EC(50), 30 nM by PerkinElmer AlphaScreen assay) and TGR5 (mean EC(50), 630 nM by time resolved-fluorescence resonance energy transfer), the first compound described so far to potently and selectively activate both BA receptors. INT-767 does not show cytotoxic effects in HepG2 cells, does not inhibit cytochrome P450 enzymes, is highly stable to phase I and II enzymatic modifications, and does not inhibit the human ether-a-go-go-related gene potassium channel. In line with its dual activity, INT-767 induces FXR-dependent lipid uptake by adipocytes, with the beneficial effect of shuttling lipids from central hepatic to peripheral fat storage, and promotes TGR5-dependent glucagon-like peptide-1 secretion by enteroendocrine cells, a validated target in the treatment of type 2 diabetes. Moreover, INT-767 treatment markedly decreases cholesterol and triglyceride levels in diabetic db/db mice and in mice rendered diabetic by streptozotocin administration. Collectively, these preclinical results indicate that INT-767 is a safe and effective modulator of FXR and TGR5-dependent pathways, suggesting potential clinical applications in the treatment of liver and metabolic diseases.

From PARP1 to TNKS2 Inhibition: A Structure-Based Approach.

Tankyrases (TNKSs) have recently gained great consideration as potential targets in Wnt/ß-catenin pathway-dependent solid tumors. Previously, we reported the 2-mercaptoquinazolin-4-one MC2050 as a micromolar PARP1 inhibitor. Here we show how the resolution of the X-ray structure of PARP1 in complex with MC2050, combined with the computational investigation of the structural differences between TNKSs and PARP1/2 active sites, provided the rationale for a structure-based drug design campaign that with a limited synthetic effort led to the discovery of the bis-quinazolinone 5 as a picomolar and selective TNKS2 inhibitor, endowed with antiproliferative effects in a colorectal cancer cell line (DLD-1) where the Wnt pathway is constitutively activated.

Btg2 enhances retinoic acid-induced differentiation by modulating histone H4 methylation and acetylation.

Retinoic acid controls hematopoietic differentiation through the transcription factor activity of its receptors. They act on specific target genes by recruiting protein complexes that deacetylate or acetylate histones and modify chromatin status. The regulation of this process is affected by histone methyltransferases, which can inhibit or activate transcription depending on their amino acid target. We show here that retinoic acid treatment of hematopoietic cells induces the expression of BTG2. Overexpression of this protein increases RARalpha transcriptional activity and the differentiation response to retinoic acid of myeloid leukemia cells and CD34+ hematopoietic progenitors. In the absence of retinoic acid, BTG2 is present in the RARalpha transcriptional complex, together with the arginine methyltransferase PRMT1 and Sin3A. Overexpressed BTG2 increases PRMT1 participation in the RARalpha protein complex on the RARbeta promoter, a target gene model, and enhances gene-specific histone H4 arginine methylation. Upon RA treatment Sin3A, BTG2, and PRMT1 detach from RARalpha and thereafter BGT2 and PRMT1 are driven to the cytoplasm. These events prime histone H4 demethylation and acetylation. Overall, our data show that BTG2 contributes to retinoic acid activity by favoring differentiation through a gene-specific modification of histone H4 arginine methylation and acetylation levels.

Dissecting the allosteric FXR modulation: a chemical biology approach using guggulsterone as a chemical tool.

Guggulsterone is a promiscuous ligand for endocrine and metabolic lipid receptors traditionally used to treat a number of diseases including diabesity, hyperlipidemia, atherosclerosis, and osteoarthritis. Although relatively weak, its activity at the farnesoid X receptor (FXR) is particularly intriguing as guggulsterone acts as an antagonist with a peculiar ability of gene selective modulation. We report here a chemical biology study with the aim to further characterize the biological action of guggulsterone at the FXR and to obtain further insights into the functional role played by noncanonical FXR binding pockets S2 and S3. Our results suggest that the FXR accessory pockets might act as potential targets for small molecules able to modulate the metabolic activation of the receptor without affecting the anti-inflammatory activity thus revealing a new approach for disclosing selective FXR modulators that might bypass potential side-effects from chronic treatments.

Exploiting Chemical Toolboxes for the Expedited Generation of Tetracyclic Quinolines as a Novel Class of PXR Agonists.

The discovery of lead compounds relies on the iterative generation of structure-activity relationship data resulting from the synthesis and biological evaluation of hit analogues. Using traditional approaches, a significant time delay may occur from compound design to results, leading to slow and expensive hit-to-lead explorations. Herein, we have exploited the use of chemical toolboxes to expedite lead discovery and optimization. In particular, the integration of flow synthesizers, automation, process analytical technologies, and computational chemistry has provided a prototype system enabling the multicomponent flow synthesis, in-line analysis, and characterization of chiral tetracyclic quinolines as a novel class of PXR agonists. Within 29 compounds, a novel template 19b (3aS,11R,11aS) was identified with an EC50 of 1.2 µM (efficacy 119%) at the PXR receptor.

Cellular retinoic acid binding protein-II expression and its potential role in skin aging.

Skin aging is an intricate biological process consisting of intrinsic and extrinsic alterations of epidermal and dermal structures. Retinoids play an important role in epidermal cell growth and differentiation and are beneficial to counteract skin aging. Cellular retinoic acid binding protein-II (CRABP-II) selectively binds all trans-retinoic acid, the most active retinoid metabolite, contributing to regulate intracytoplasmic retinoid trafficking and keratinocyte differentiation. Immunohistochemistry revealed a reduced epidermal and dermal CRABP-II expression in aged human and mouse skin. To better clarify the role of CRABP-II, we investigated age-related skin changes in CRABP-II knock-out mice. We documented an early reduction of keratinocyte layers, proliferation and differentiation rate, dermal and hypodermal thickness, pilosebaceous units and dermal vascularity in CRABP-II knock-out compared with wild-type mice. Ultrastructural investigation documented reduced number and secretion of epidermal lamellar bodies in CRABP-II knock-out compared with wild-type mice. Cultured CRABP-II knock-out-derived dermal fibroblasts proliferated less and showed reduced levels of TGF-ß signal-related genes, Col1A1, Col1A2, and increased MMP2 transcripts compared with those from wild-type. Our data strongly support the hypothesis that a reduction of CRABP-II expression accelerates and promotes skin aging, and suggest CRABP-II as a novel target to improve the efficacy of retinoid-mediated anti-aging therapies.