Small molecule approaches to targeting RNA.

The development of innovative methodologies to identify RNA binders has attracted enormous attention in chemical biology and drug discovery. Although antibiotics targeting bacterial ribosomal RNA have been on the market for decades, the renewed interest in RNA targeting reflects the need to better understand complex intracellular processes involving RNA. In this context, small molecules are privileged tools used to explore the biological functions of RNA and to validate RNAs as therapeutic targets, and they eventually are to become new drugs. Despite recent progress, the rational design of specific RNA binders requires a better understanding of the interactions which occur with the RNA target to reach the desired biological response. In this Review, we discuss the challenges to approaching this underexplored chemical space, together with recent strategies to bind, interact and affect biologically relevant RNAs.

Identification of a Dual Autophagy and REV-ERB Inhibitor with in Vivo Anticancer Efficacy.

The autophagy process appears as a promising target for anticancer interventions. Chloroquine (CQ) and its derivative hydroxychloroquine (HCQ) are the only FDA-approved autophagy flux inhibitors. Although diverse anticancer clinical trials are providing encouraging results, several limitations associated with the need of high dosage and long-term administration of these autophagy inhibitors are also emerging. We showed that the inhibition of REV-ERB, a nuclear receptor regulating circadian rhythm and metabolism, enhances CQ-mediated cancer cell death and identified a class of dual inhibitors of autophagy and REV-ERB displaying an in vitro anticancer activity against diverse tumor cells greatly higher than CQ. Herein, we describe our lead optimization strategy that led to the identification of compound 24 as a dual autophagy and REV-ERB inhibitor, showing improved potency in blocking autophagy, enhanced toxicity against cancer cells, optimal drug-like properties, and efficacy in a mouse xenograft model of melanoma as a single anticancer agent.

Autophagy and cancer drug resistance in dialogue: Pre-clinical and clinical evidence.

The emergence of drug resistance is a major challenge for oncologists. Resistance can be categorized as acquired or intrinsic; the alteration of several biological mechanisms contributes to both intrinsic and acquired resistance. Macroautophagy/autophagy is the primary process in eukaryotes for the degradation of macromolecules and organelles. This process is critical in maintaining cellular homeostasis. Given its function as either a pro-survival or a pro-death phenomenon, autophagy has a complex physio-pathological role. In some circumstances, autophagy can confer chemoresistance and promote cell survival, whereas in others it can promote chemosensitivity and contribute to cell death. The role of autophagy in the modulation of cancer drug resistance reflects its impact on apoptosis and metastasis. The regulation of autophagy in cancer is mediated by various factors including AMP-activated protein kinase (AMPK), MAPK, phosphoinositide 3-kinase (PI3K)-AKT, BECN1 and ATG proteins. Non-coding RNAs are among the main regulators of autophagy, e.g., via the modulation of chemoresistance pathways. Due to the significant contribution of autophagy in cancer drug resistance, small molecule modulators and natural compounds targeting autophagy have been introduced to alter the response of cancer cells to chemotherapy. Furthermore, nanotherapeutic approaches based on autophagy regulation have been introduced in pre-clinical cancer therapy. In this review we consider the potential for using autophagy regulators for the clinical treatment of malignancies.

MYC-Associated Factor MAX is a Regulator of the Circadian Clock.

The circadian transcriptional network is based on a competition between transcriptional activator and repressor complexes regulating the rhythmic expression of clock-controlled genes. We show here that the MYC-associated factor X, MAX, plays a repressive role in this network and operates through a MYC-independent binding to E-box-containing regulatory regions within the promoters of circadian BMAL1 targets. We further show that this "clock" function of MAX is required for maintaining a proper circadian rhythm and that MAX and BMAL1 contribute to two temporally alternating transcriptional complexes on clock-regulated promoters. We also identified MAX network transcriptional repressor, MNT, as a fundamental partner of MAX-mediated circadian regulation. Collectively, our data indicate that MAX regulates clock gene expression and contributes to keeping the balance between positive and negative elements of the molecular clock machinery.

Disulfide-Mediated Bioconjugation: Disulfide Formation and Restructuring on the Surface of Nanomanufactured (Microfluidics) Nanoparticles.

This study is about (1) nanomanufacturing (focusing on microfluidic-assisted nanoprecipitation), (2) advanced colloid characterization (focusing on field flow fractionation), and (3) the possible restructuring of surface disulfides. Disulfides are dynamic and exchangeable groups, and here we specifically focus, first, on their use to introduce biofunctional groups and, second, on their re-organization, which may lead to variable surface chemistries and uncontrolled cell interactions. The particles were obtained via microfluidic-assisted (flow-focused) nanoprecipitation of poly(ethylene glycol)-b-poly(ε-caprolactone) bearing or not a 2-pyridyl disulfide (PDS) terminal group, which quantitatively exchanges with thiols in solution. In this study, we have paid specific attention to size characterization, thereby also demonstrating the limitations of dynamic light scattering (DLS) as a stand-alone technique. By using asymmetric flow field flow fractionation coupled with DLS, static light scattering (SLS), and refractive index detectors, we show that relatively small amounts of >100 nm aggregates (cryogenic transmission electron microscopy and SLS/DLS comparison suggesting them to be wormlike micelles) dominated the stand-alone DLS results, whereas the "real" size distributions picked <50 nm. Our key result is that the kinetics of the conjugation based on PDS-thiol exchange was controlled by the thiol pKa, and this also determined the rate of the exchange between the resulting disulfides and glutathione (GSH). In particular, more acidic thiols (e.g., peptides, where a cysteine is flanked by cationic residues) react faster with PDS, but their disulfides hardly exchange with GSH; the reverse applies to thiols with a higher pKa. Disulfides that resist against restructuring via thiol-disulfide exchange allow for a stable bioconjugation, although they may be bad news for payload release under reducing conditions. However, experiments of both thiol release and nanoparticles uptake in cells (HCT116) show that also the disulfides formed from less-acidic and, therefore, less-reactive, and more exchangeable thiols were stable for at least a few hours even in a GSH-rich (10 mM) environment; this suggests a sufficiently long stability of surface groups to achieve, for example, a cell-targeting effect.

A broad-spectrum antibiotic, DCAP, reduces uropathogenic Escherichia coli infection and enhances vorinostat anticancer activity by modulating autophagy.

The cellular recycling pathway of autophagy plays a fundamental role in adaptive responses to nutrient deprivation and other forms of stress under physiological and pathological conditions. However, autophagy can also be a double-edge sword during certain bacterial infections (such as urinary tract infections) and in cancer, where it can be hijacked by the pathogens and cancer cells, respectively, to promote their own survival. Thus, autophagy modulation can potentially have multiple effects in multiple contexts and this property can be leveraged to improve outcomes. In this report, we identify that a broad-spectrum antibiotic, 2-((3-(3, 6-dichloro-9H-carbazol-9-yl)-2-hydroxypropyl) amino)-2-(hydroxymethyl) propane-1, 3-diol (DCAP) modulates autophagy. We employed combined biochemical, fluorescence microscopy and correlative light electron microscopy approaches to demonstrate that DCAP treatment blocks autophagy at the late stages by preventing autophagolysosome maturation and interrupting the autophagic flux. We further show that, DCAP significantly reduces UPEC infection in urinary tract epithelial cells via inhibition of autophagy. Finally, we reveal that DCAP enhances the anticancer activity of the histone acetyltransferase (HDAC) inhibitor, vorinostat, which has been reported to increase susceptibility to bacterial infections as a common adverse effect. Collectively, our data support the concept that DCAP represents a valuable chemical scaffold for the development of an innovative class of bactericidal autophagy inhibitors for treatment of urinary tract infections and/or for adjuvant therapy in cancer treatment.

Autophagy is essential for maintaining the growth of a human (mini-)organ: Evidence from scalp hair follicle organ culture.

Autophagy plays a crucial role in health and disease, regulating central cellular processes such as adaptive stress responses, differentiation, tissue development, and homeostasis. However, the role of autophagy in human physiology is poorly understood, highlighting a need for a model human organ system to assess the efficacy and safety of strategies to therapeutically modulate autophagy. As a complete, cyclically remodelled (mini-)organ, the organ culture of human scalp hair follicles (HFs), which, after massive growth (anagen), spontaneously enter into an apoptosis-driven organ involution (catagen) process, may provide such a model. Here, we reveal that in anagen, hair matrix keratinocytes (MKs) of organ-cultured HFs exhibit an active autophagic flux, as documented by evaluation of endogenous lipidated Light Chain 3B (LC3B) and sequestosome 1 (SQSTM1/p62) proteins and the ultrastructural visualization of autophagosomes at all stages of the autophagy process. This autophagic flux is altered during catagen, and genetic inhibition of autophagy promotes catagen development. Conversely, an anti-hair loss product markedly enhances intrafollicular autophagy, leading to anagen prolongation. Collectively, our data reveal a novel role of autophagy in human hair growth. Moreover, we show that organ-cultured scalp HFs are an excellent preclinical research model for exploring the role of autophagy in human tissue physiology and for evaluating the efficacy and tissue toxicity of candidate autophagy-modulatory agents in a living human (mini-)organ.

Circadian clock: Time for novel anticancer strategies?

Disruption of the circadian clock is associated with a variety of human pathologies, including cancer. Rather than being a mere consequence of a global changes associated with the cancer cell transcriptome, the aberrant clock gene expression observed in many tumors may serve for cancer cell survival. This scenario suggests the provocative hypothesis that pharmacological modulation of clock-related proteins may be suitable as an effective anticancer strategy. In this review, we focus on the functions of the druggable circadian nuclear receptors, REV-ERBα and REV-ERBß, in cancer cell survival and describe the potential development of small molecule compounds that modulate REV-ERB activity as novel anticancer therapeutics. In addition, we debate the use of circadian rhythm-based synthetic lethal approaches to identify yet unexplored anticancer strategies.

Synthesis and in Vitro Anticancer Activity of the First Class of Dual Inhibitors of REV-ERBß and Autophagy.

Autophagy inhibition is emerging as a promising anticancer strategy. We recently reported that the circadian nuclear receptor REV-ERBß plays an unexpected role in sustaining cancer cell survival when the autophagy flux is compromised. We also identified 4-[[[1-(2-fluorophenyl)cyclopentyl]amino]methyl]-2-[(4-methylpiperazin-1-yl)methyl]phenol, 1 (ARN5187), as a novel dual inhibitor of REV-ERBß and autophagy. 1 had improved cytotoxicity against BT-474 breast cancer cells compared to chloroquine, a clinically relevant autophagy inhibitor. Here, we present the results of structure-activity studies, based around 1, that disclose the first class of dual inhibitors of REV-ERBß and autophagy. This study led to identification of 18 and 28, which were more effective REV-ERBß antagonists than 1 and were more cytotoxic to BT-474. The combination of optimal chemical and structural moieties of these analogs generated 30, which elicited 15-fold greater REV-ERBß inhibitory and cytotoxic activities compared to 1. Furthermore, 30 induced death in a panel of tumor cell lines at doses 5-50 times lower than an equitoxic amount of chloroquine but did not affect the viability of normal mammary epithelial cells.

Methamphetamine accelerates cellular senescence through stimulation of de novo ceramide biosynthesis.

Methamphetamine is a highly addictive psychostimulant that causes profound damage to the brain and other body organs. Post mortem studies of human tissues have linked the use of this drug to diseases associated with aging, such as coronary atherosclerosis and pulmonary fibrosis, but the molecular mechanism underlying these findings remains unknown. Here we used functional lipidomics and transcriptomics experiments to study abnormalities in lipid metabolism in select regions of the brain and, to a greater extent, peripheral organs and tissues of rats that self-administered methamphetamine. Experiments in various cellular models (primary mouse fibroblasts and myotubes) allowed us to investigate the molecular mechanisms of systemic inflammation and cellular aging related to methamphetamine abuse. We report now that methamphetamine accelerates cellular senescence and activates transcription of genes involved in cell-cycle control and inflammation by stimulating production of the sphingolipid messenger ceramide. This pathogenic cascade is triggered by reactive oxygen species, likely generated through methamphetamine metabolism via cytochrome P450, and involves the recruitment of nuclear factor-κB (NF-κB) to induce expression of enzymes in the de novo pathway of ceramide biosynthesis. Inhibitors of NF-κB signaling and ceramide formation prevent methamphetamine-induced senescence and systemic inflammation in rats self-administering the drug, attenuating their health deterioration. The results suggest new therapeutic strategies to reduce the adverse consequences of methamphetamine abuse and improve effectiveness of abstinence treatments.

Lysosomotropic REV-ERB antagonism: A metabolic connection between circadian rhythm and autophagy may tell cancer cells "it's time to die".

The discovery that inhibition of a circadian regulator enhances autophagy-dependent cancer cell death reveals potential avenues for the development of new multifunctional anticancer agents. Further studies may elucidate novel crosstalk between circadian rhythm, metabolism, and autophagy that determines cancer cell viability.

The peripheral clock regulates human pigmentation.

Although the regulation of pigmentation is well characterized, it remains unclear whether cell-autonomous controls regulate the cyclic on-off switching of pigmentation in the hair follicle (HF). As human HFs and epidermal melanocytes express clock genes and proteins, and given that core clock genes (PER1, BMAL1) modulate human HF cycling, we investigated whether peripheral clock activity influences human HF pigmentation. We found that silencing BMAL1 or PER1 in human HFs increased HF melanin content. Furthermore, tyrosinase expression and activity, as well as TYRP1 and TYRP2 mRNA levels, gp100 protein expression, melanocyte dendricity, and the number gp100+ HF melanocytes, were all significantly increased in BMAL1 and/or PER1-silenced HFs. BMAL1 or PER1 silencing also increased epidermal melanin content, gp100 protein expression, and tyrosinase activity in human skin. These effects reflect direct modulation of melanocytes, as BMAL1 and/or PER1 silencing in isolated melanocytes increased tyrosinase activity and TYRP1/2 expression. Mechanistically, BMAL1 knockdown reduces PER1 transcription, and PER1 silencing induces phosphorylation of the master regulator of melanogenesis, microphthalmia-associated transcription factor, thus stimulating human melanogenesis and melanocyte activity in situ and in vitro. Therefore, the molecular clock operates as a cell-autonomous modulator of human pigmentation and may be targeted for future therapeutic strategies.

Circadian control of fatty acid elongation by SIRT1 protein-mediated deacetylation of acetyl-coenzyme A synthetase 1.

The circadian clock regulates a wide range of physiological and metabolic processes, and its disruption leads to metabolic disorders such as diabetes and obesity. Accumulating evidence reveals that the circadian clock regulates levels of metabolites that, in turn, may regulate the clock. Here we demonstrate that the circadian clock regulates the intracellular levels of acetyl-CoA by modulating the enzymatic activity of acetyl-CoA Synthetase 1 (AceCS1). Acetylation of AceCS1 controls the activity of the enzyme. We show that acetylation of AceCS1 is cyclic and that its rhythmicity requires a functional circadian clock and the NAD(+)-dependent deacetylase SIRT1. Cyclic acetylation of AceCS1 contributes to the rhythmicity of acetyl-CoA levels both in vivo and in cultured cells. Down-regulation of AceCS1 causes a significant decrease in the cellular acetyl-CoA pool, leading to reduction in circadian changes in fatty acid elongation. Thus, a nontranscriptional, enzymatic loop is governed by the circadian clock to control acetyl-CoA levels and fatty acid synthesis.

A meeting of two chronobiological systems: circadian proteins Period1 and BMAL1 modulate the human hair cycle clock.

The hair follicle (HF) is a continuously remodeled mini organ that cycles between growth (anagen), regression (catagen), and relative quiescence (telogen). As the anagen-to-catagen transformation of microdissected human scalp HFs can be observed in organ culture, it permits the study of the unknown controls of autonomous, rhythmic tissue remodeling of the HF, which intersects developmental, chronobiological, and growth-regulatory mechanisms. The hypothesis that the peripheral clock system is involved in hair cycle control, i.e., the anagen-to-catagen transformation, was tested. Here we show that in the absence of central clock influences, isolated, organ-cultured human HFs show circadian changes in the gene and protein expression of core clock genes (CLOCK, BMAL1, and Period1) and clock-controlled genes (c-Myc, NR1D1, and CDKN1A), with Period1 expression being hair cycle dependent. Knockdown of either BMAL1 or Period1 in human anagen HFs significantly prolonged anagen. This provides evidence that peripheral core clock genes modulate human HF cycling and are an integral component of the human hair cycle clock. Specifically, our study identifies BMAL1 and Period1 as potential therapeutic targets for modulating human hair growth.

Circadian clock regulates the host response to Salmonella.

Organisms adapt to day-night cycles through highly specialized circadian machinery, whose molecular components anticipate and drive changes in organism behavior and metabolism. Although many effectors of the immune system are known to follow daily oscillations, the role of the circadian clock in the immune response to acute infections is not understood. Here we show that the circadian clock modulates the inflammatory response during acute infection with the pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium). Mice infected with S. Typhimurium were colonized to higher levels and developed a higher proinflammatory response during the early rest period for mice, compared with other times of the day. We also demonstrate that a functional clock is required for optimal S. Typhimurium colonization and maximal induction of several proinflammatory genes. These findings point to a clock-regulated mechanism of activation of the immune response against an enteric pathogen and may suggest potential therapeutic strategies for chronopharmacologic interventions.

Physical association of the WC-1 photoreceptor and the histone acetyltransferase NGF-1 is required for blue light signal transduction in Neurospora crassa.

In Neurospora crassa and other filamentous fungi, light-dependent-specific phenomena are regulated by transcription factors WC-1 and WC-2. In addition to its transcriptional activity, WC-1 is able to directly sense light stimuli through a LOV sensor domain. Its location in the nucleus and heterodimerization with WC-2, together with the presence of a zinc-finger DNA-binding domain and an environmental sensor domain, all resemble the functional evolutionary architecture adopted by vertebrate nuclear receptors (NRs). Here we describe a scenario in which WC-1 represents a functional orthologue of NRs and acts through association with the chromatin-modifying coactivator NGF-1, which encodes a homologue of the yeast Gcn5p acetyltransferase. To support this view, we show a direct association between WC-1 and NGF-1 that depends on a WC-1 region containing a conserved functional LXXLL motif, a signature previously described as being an exclusive feature of NR/coactivator interaction. Our data suggest that a WC-1/NGF-1 complex is preassembled in the dark on light-inducible promoters and that, after exposure to light stimulation, NGF-1-associated HAT activity leads to histone H3 acetylation and transcriptional activation. Finally, we provide evidence for a NGF-1-independent acetylated form of WC-1. Overall our data indicate that Neurospora and higher eukaryotes share a common mechanism for the signal transduction of environmental stimuli.

PER2 controls lipid metabolism by direct regulation of PPARγ.

Accumulating evidence highlights intriguing interplays between circadian and metabolic pathways. We show that PER2 directly and specifically represses PPARγ, a nuclear receptor critical in adipogenesis, insulin sensitivity, and inflammatory response. PER2-deficient mice display altered lipid metabolism with drastic reduction of total triacylglycerol and nonesterified fatty acids. PER2 exerts its inhibitory function by blocking PPARγ recruitment to target promoters and thereby transcriptional activation. Whole-genome microarray profiling demonstrates that PER2 dictates the specificity of PPARγ transcriptional activity. Indeed, lack of PER2 results in enhanced adipocyte differentiation of cultured fibroblasts. PER2 targets S112 in PPARγ, a residue whose mutation has been associated with altered lipid metabolism. Lipidomic profiling demonstrates that PER2 is necessary for normal lipid metabolism in white adipocyte tissue. Our findings support a scenario in which PER2 controls the proadipogenic activity of PPARγ by operating as its natural modulator, thereby revealing potential avenues of pharmacological and therapeutic intervention.

Chromatin remodeling, metabolism and circadian clocks: the interplay of CLOCK and SIRT1.

Circadian rhythms govern a wide variety of physiological and metabolic functions in almost all organisms. These are controlled by the circadian clock machinery, which is mostly based on transcriptional-translational feedback loops. Importantly, 10-15% of the mammalian transcripts oscillate in a circadian manner. The complex program of gene expression that characterizes circadian physiology is possible through dynamic changes in chromatin transitions. These remodeling events are therefore of great importance to insure the proper timing and extent of circadian regulation. Recent advances in the field have revealed unexpected links between circadian regulators, chromatin remodeling and cellular metabolism. Specifically, the central clock protein CLOCK has HAT enzymatic properties. It directs acetylation of histone H3 and of its dimerization partner BMAL1 at K537, an event essential for circadian function. In addition, the HDAC activity of the NAD(+)-dependent SIRT1 enzyme is regulated in a circadian manner. It has been proposed that SIRT1 functions as an enzymatic rheostat of circadian function, transducing signals originated by cellular metabolites to the circadian clock. Thus, a specialized program of chromatin remodeling appears to be at the core of the circadian machinery.

The NAD+-dependent deacetylase SIRT1 modulates CLOCK-mediated chromatin remodeling and circadian control.

Circadian rhythms govern a large array of metabolic and physiological functions. The central clock protein CLOCK has HAT properties. It directs acetylation of histone H3 and of its dimerization partner BMAL1 at Lys537, an event essential for circadian function. We show that the HDAC activity of the NAD(+)-dependent SIRT1 enzyme is regulated in a circadian manner, correlating with rhythmic acetylation of BMAL1 and H3 Lys9/Lys14 at circadian promoters. SIRT1 associates with CLOCK and is recruited to the CLOCK:BMAL1 chromatin complex at circadian promoters. Genetic ablation of the Sirt1 gene or pharmacological inhibition of SIRT1 activity lead to disturbances in the circadian cycle and in the acetylation of H3 and BMAL1. Finally, using liver-specific SIRT1 mutant mice we show that SIRT1 contributes to circadian control in vivo. We propose that SIRT1 functions as an enzymatic rheostat of circadian function, transducing signals originated by cellular metabolites to the circadian clock.

CLOCK-mediated acetylation of BMAL1 controls circadian function.

Regulation of circadian physiology relies on the interplay of interconnected transcriptional-translational feedback loops. The CLOCK-BMAL1 complex activates clock-controlled genes, including cryptochromes (Crys), the products of which act as repressors by interacting directly with CLOCK-BMAL1. We have demonstrated that CLOCK possesses intrinsic histone acetyltransferase activity and that this enzymatic function contributes to chromatin-remodelling events implicated in circadian control of gene expression. Here we show that CLOCK also acetylates a non-histone substrate: its own partner, BMAL1, is specifically acetylated on a unique, highly conserved Lys 537 residue. BMAL1 undergoes rhythmic acetylation in mouse liver, with a timing that parallels the downregulation of circadian transcription of clock-controlled genes. BMAL1 acetylation facilitates recruitment of CRY1 to CLOCK-BMAL1, thereby promoting transcriptional repression. Importantly, ectopic expression of a K537R-mutated BMAL1 is not able to rescue circadian rhythmicity in a cellular model of peripheral clock. These findings reveal that the enzymatic interplay between two clock core components is crucial for the circadian machinery.