Structure-based mechanism of riboregulation of the metabolic enzyme SHMT1.

RNA can directly control protein activity in a process called riboregulation; only a few mechanisms of riboregulation have been described in detail, none of which have been characterized on structural grounds. Here, we present a comprehensive structural, functional, and phylogenetic analysis of riboregulation of cytosolic serine hydroxymethyltransferase (SHMT1), the enzyme interconverting serine and glycine in one-carbon metabolism. We have determined the cryoelectron microscopy (cryo-EM) structure of human SHMT1 in its free- and RNA-bound states, and we show that the RNA modulator competes with polyglutamylated folates and acts as an allosteric switch, selectively altering the enzyme's reactivity vs. serine. In addition, we identify the tetrameric assembly and a flap structural motif as key structural elements necessary for binding of RNA to eukaryotic SHMT1. The results presented here suggest that riboregulation may have played a role in evolution of eukaryotic SHMT1 and in compartmentalization of one-carbon metabolism. Our findings provide insights for RNA-based therapeutic strategies targeting this cancer-linked metabolic pathway.

A host-directed oxadiazole compound potentiates antituberculosis treatment via zinc poisoning in human macrophages and in a mouse model of infection.

Antituberculosis drugs, mostly developed over 60 years ago, combined with a poorly effective vaccine, have failed to eradicate tuberculosis. More worryingly, multiresistant strains of Mycobacterium tuberculosis (MTB) are constantly emerging. Innovative strategies are thus urgently needed to improve tuberculosis treatment. Recently, host-directed therapy has emerged as a promising strategy to be used in adjunct with existing or future antibiotics, by improving innate immunity or limiting immunopathology. Here, using high-content imaging, we identified novel 1,2,4-oxadiazole-based compounds, which allow human macrophages to control MTB replication. Genome-wide gene expression analysis revealed that these molecules induced zinc remobilization inside cells, resulting in bacterial zinc intoxication. More importantly, we also demonstrated that, upon treatment with these novel compounds, MTB became even more sensitive to antituberculosis drugs, in vitro and in vivo, in a mouse model of tuberculosis. Manipulation of heavy metal homeostasis holds thus great promise to be exploited to develop host-directed therapeutic interventions.

The α-tubulin acetyltransferase ATAT1: structure, cellular functions, and its emerging role in human diseases.

The acetylation of α-tubulin on lysine 40 is a well-studied post-translational modification which has been associated with the presence of long-lived stable microtubules that are more resistant to mechanical breakdown. The discovery of α-tubulin acetyltransferase 1 (ATAT1), the enzyme responsible for lysine 40 acetylation on α-tubulin in a wide range of species, including protists, nematodes, and mammals, dates to about a decade ago. However, the role of ATAT1 in different cellular activities and molecular pathways has been only recently disclosed. This review comprehensively summarizes the most recent knowledge on ATAT1 structure and substrate binding and analyses the involvement of ATAT1 in a variety of cellular processes such as cell motility, mitosis, cytoskeletal organization, and intracellular trafficking. Finally, the review highlights ATAT1 emerging roles in human diseases and discusses ATAT1 potential enzymatic and non-enzymatic roles and the current efforts in developing ATAT1 inhibitors.

Activation and inhibition of sirtuins: From bench to bedside.

The sirtuin family comprises seven NAD+-dependent enzymes which catalyze protein lysine deacylation and mono ADP-ribosylation. Sirtuins act as central regulators of genomic stability and gene expression and control key processes, including energetic metabolism, cell cycle, differentiation, apoptosis, and aging. As a result, all sirtuins play critical roles in cellular homeostasis and organism wellness, and their dysregulation has been linked to metabolic, cardiovascular, and neurological diseases. Furthermore, sirtuins have shown dichotomous roles in cancer, acting as context-dependent tumor suppressors or promoters. Given their central role in different cellular processes, sirtuins have attracted increasing research interest aimed at developing both activators and inhibitors. Indeed, sirtuin modulation may have therapeutic effects in many age-related diseases, including diabetes, cardiovascular and neurodegenerative disorders, and cancer. Moreover, isoform selective modulators may increase our knowledge of sirtuin biology and aid to develop better therapies. Through this review, we provide critical insights into sirtuin pharmacology and illustrate their enzymatic activities and biological functions. Furthermore, we outline the most relevant sirtuin modulators in terms of their modes of action, structure-activity relationships, pharmacological effects, and clinical applications.

Targeting of H19/cell adhesion molecules circuitry by GSK-J4 epidrug inhibits metastatic progression in prostate cancer.

BACKGROUND: About 30% of Prostate cancer (PCa) patients progress to metastatic PCa that remains largely incurable. This evidence underlines the need for the development of innovative therapies. In this direction, the potential research focus might be on long non-coding RNAs (lncRNAs) like H19, which serve critical biological functions and show significant dysregulation in cancer. Previously, we showed a transcriptional down-regulation of H19 under combined pro-tumoral estrogen and hypoxia treatment in PCa cells that, in turn, induced both E-cadherin and ß4 integrin expression. H19, indeed, acts as transcriptional repressor of cell adhesion molecules affecting the PCa metastatic properties. Here, we investigated the role of H19/cell adhesion molecules circuitry on in vivo PCa experimental tumor growth and metastatic dissemination models. METHODS: H19 was silenced in luciferase-positive PC-3 and 22Rv1 cells and in vitro effect was evaluated by gene expression, proliferation and invasion assays before and after treatment with the histone lysine demethylase inhibitor, GSK-J4. In vivo tumor growth and metastasis dissemination, in the presence or absence of GSK-J4, were analyzed in two models of human tumor in immunodeficient mice by in vivo bioluminescent imaging and immunohistochemistry (IHC) on explanted tissues. Organotypic Slice Cultures (OSCs) from fresh PCa-explant were used as ex vivo model to test GSK-J4 effects. RESULTS: H19 silencing in both PC-3 and 22Rv1 cells increased: i) E-cadherin and ß4 integrin expression as well as proliferation and invasion, ii) in vivo tumor growth, and iii) metastasis formation at bone, lung, and liver. Of note, treatment with GSK-J4 reduced lesions. In parallel, GSK-J4 efficiently induced cell death in PCa-derived OSCs. CONCLUSIONS: Our findings underscore the potential of the H19/cell adhesion molecules circuitry as a targeted approach in PCa treatment. Modulating this interaction has proven effective in inhibiting tumor growth and metastasis, presenting a logical foundation for targeted therapy.

Transcriptomic and genomic studies classify NKL54 as a histone deacetylase inhibitor with indirect influence on MEF2-dependent transcription.

In leiomyosarcoma class IIa HDACs (histone deacetylases) bind MEF2 and convert these transcription factors into repressors to sustain proliferation. Disruption of this complex with small molecules should antagonize cancer growth. NKL54, a PAOA (pimeloylanilide o-aminoanilide) derivative, binds a hydrophobic groove of MEF2, which is used as a docking site by class IIa HDACs. However, NKL54 could also act as HDAC inhibitor (HDACI). Therefore, it is unclear which activity is predominant. Here, we show that NKL54 and similar derivatives are unable to release MEF2 from binding to class IIa HDACs. Comparative transcriptomic analysis classifies these molecules as HDACIs strongly related to SAHA/vorinostat. Low expressed genes are upregulated by HDACIs, while abundant genes are repressed. This transcriptional resetting correlates with a reorganization of H3K27 acetylation around the transcription start site (TSS). Among the upregulated genes there are several BH3-only family members, thus explaining the induction of apoptosis. Moreover, NKL54 triggers the upregulation of MEF2 and the downregulation of class IIa HDACs. NKL54 also increases the binding of MEF2D to promoters of genes that are upregulated after treatment. In summary, although NKL54 cannot outcompete MEF2 from binding to class IIa HDACs, it supports MEF2-dependent transcription through several actions, including potentiation of chromatin binding.

Hydroxamate-based compounds are potent inhibitors of Toxoplasma gondii HDAC biological activity.

Toxoplasmosis is a critical health issue for immune-deficient individuals and the offspring of newly infected mothers. It is caused by a unicellular intracellular parasite called Toxoplasma gondii that is found worldwide. Although efficient drugs are commonly used to treat toxoplasmosis, serious adverse events are common. Therefore, new compounds with potent anti-T. gondii activity are needed to provide better suited treatments. We have tested compounds designed to target specifically histone deacetylase enzymes. Among the 55 compounds tested, we identified three compounds showing a concentration of drug required for 50% inhibition (IC50) in the low 100 nM range with a selectivity index of more than 100. These compounds are not only active at inhibiting the growth of the parasite in vitro but also at preventing some of the consequences of the acute disease in vivo. Two of these hydroxamate based compound also induce a hyper-acetylation of the parasite histones while the parasitic acetylated tubulin level remains unchanged. These findings suggest that the enzymes regulating histone acetylation are potent therapeutic targets for the treatment of acute toxoplasmosis.

Simultaneous administration of EZH2 and BET inhibitors inhibits proliferation and clonogenic ability of metastatic prostate cancer cells.

Androgen deprivation therapy (ADT) is a common treatment for recurrent prostate cancer (PC). However, after a certain period of responsiveness, ADT resistance occurs virtually in all patients and the disease progresses to lethal metastatic castration-resistant prostate cancer (mCRPC). Aberrant expression and function of the epigenetic modifiers EZH2 and BET over activates c-myc, an oncogenic transcription factor critically contributing to mCRPC. In the present work, we tested, for the first time, the combination of an EZH2 inhibitor with a BET inhibitor in metastatic PC cells. The combination outperformed single drugs in inhibiting cell viability, cell proliferation and clonogenic ability, and concomitantly reduced both c-myc and NF-kB expression. Although these promising results will warrant further in vivo validation, they represent the first step to establishing the rationale that the proposed combination might be suitable for mCRPC treatment, by exploiting molecular targets different from androgen receptor.

Antihistamines, phenothiazine-based antipsychotics, and tricyclic antidepressants potently activate pharmacologically relevant human carbonic anhydrase isoforms II and VII.

Carbonic anhydrases (CAs) are important regulators of pH homeostasis and participate in many physiological and pathological processes. CA activators (CAAs) are becoming increasingly important in the biomedical field since enhancing CA activity may have beneficial effects at neurological level. Here, we investigate selected antihistamines, phenothiazine-based antipsychotics, and tricyclic antidepressants (TCAs) as potential activators of human CAs I, II, IV, and VII. Our findings indicate that these compounds are more effective at activating hCA II and VII compared to hCA I and IV. Overall, hCA VII was the most efficiently activated isoform, particularly by phenothiazines and TCAs. This is especially relevant since hCA VII is the most abundant isoform in the central nervous system (CNS) and is implicated in neuronal signalling and bicarbonate balance regulation. This study offers additional insights into the pharmacological profiles of clinically employed drugs and sets the ground for the development of novel optimised CAAs.

Metabolic Rewiring by Loss of Sirt5 Promotes Kras-Induced Pancreatic Cancer Progression.

BACKGROUND & AIMS: SIRT5 plays pleiotropic roles via post-translational modifications, serving as a tumor suppressor, or an oncogene, in different tumors. However, the role SIRT5 plays in the initiation and progression of pancreatic ductal adenocarcinoma (PDAC) remains unknown. METHODS: Published datasets and tissue arrays with SIRT5 staining were used to investigate the clinical relevance of SIRT5 in PDAC. Furthermore, to define the role of SIRT5 in the carcinogenesis of PDAC, we generated autochthonous mouse models with conditional Sirt5 knockout. Moreover, to examine the mechanistic role of SIRT5 in PDAC carcinogenesis, SIRT5 was knocked down in PDAC cell lines and organoids, followed by metabolomics and proteomics studies. A novel SIRT5 activator was used for therapeutic studies in organoids and patient-derived xenografts. RESULTS: SIRT5 expression negatively regulated tumor cell proliferation and correlated with a favorable prognosis in patients with PDAC. Genetic ablation of Sirt5 in PDAC mouse models promoted acinar-to-ductal metaplasia, precursor lesions, and pancreatic tumorigenesis, resulting in poor survival. Mechanistically, SIRT5 loss enhanced glutamine and glutathione metabolism via acetylation-mediated activation of GOT1. A selective SIRT5 activator, MC3138, phenocopied the effects of SIRT5 overexpression and exhibited antitumor effects on human PDAC cells. MC3138 also diminished nucleotide pools, sensitizing human PDAC cell lines, organoids, and patient-derived xenografts to gemcitabine. CONCLUSIONS: Collectively, we identify SIRT5 as a key tumor suppressor in PDAC, whose loss promotes tumorigenesis through increased noncanonic use of glutamine via GOT1, and that SIRT5 activation is a novel therapeutic strategy to target PDAC.

Biochemical Functions and Clinical Characterizations of the Sirtuins in Diabetes-Induced Retinal Pathologies.

Diabetic retinopathy (DR) is undoubtedly one of the most prominent causes of blindness worldwide. This pathology is the most frequent microvascular complication arising from diabetes, and its incidence is increasing at a constant pace. To date, the insurgence of DR is thought to be the consequence of the intricate complex of relations connecting inflammation, the generation of free oxygen species, and the consequent oxidative stress determined by protracted hyperglycemia. The sirtuin (SIRT) family comprises 7 histone and non-histone protein deacetylases and mono (ADP-ribosyl) transferases regulating different processes, including metabolism, senescence, DNA maintenance, and cell cycle regulation. These enzymes are involved in the development of various diseases such as neurodegeneration, cardiovascular pathologies, metabolic disorders, and cancer. SIRT1, 3, 5, and 6 are key enzymes in DR since they modulate glucose metabolism, insulin sensitivity, and inflammation. Currently, indirect and direct activators of SIRTs (such as antagomir, glycyrrhizin, and resveratrol) are being developed to modulate the inflammation response arising during DR. In this review, we aim to illustrate the most important inflammatory and metabolic pathways connecting SIRT activity to DR, and to describe the most relevant SIRT activators that might be proposed as new therapeutics to treat DR.

Anti-influenza A virus activity and structure-activity relationship of a series of nitrobenzoxadiazole derivatives.

Influenza viruses represent a major threat to human health and are responsible for seasonal epidemics, along with pandemics. Currently, few therapeutic options are available, with most drugs being at risk of the insurgence of resistant strains. Hence, novel approaches targeting less explored pathways are urgently needed. In this work, we assayed a library of nitrobenzoxadiazole derivatives against the influenza virus A/Puerto Rico/8/34 H1N1 (PR8) strain. We identified three promising 4-thioether substituted nitrobenzoxadiazoles (12, 17, and 25) that were able to inhibit viral replication at low micromolar concentrations in two different infected cell lines using a haemagglutination assay. We further assessed these molecules using an In-Cell Western assay, which confirmed their potency in the low micromolar range. Among the three molecules, 12 and 25 displayed the most favourable profile of activity and selectivity and were selected as hit compounds for future optimisation studies.

Epigenetic polypharmacology: A new frontier for epi-drug discovery.

Recently, despite the great success achieved by the so-called "magic bullets" in the treatment of different diseases through a marked and specific interaction with the target of interest, the pharmacological research is moving toward the development of "molecular network active compounds," embracing the related polypharmacology approach. This strategy was born to overcome the main limitations of the single target therapy leading to a superior therapeutic effect, a decrease of adverse reactions, and a reduction of potential mechanism(s) of drug resistance caused by robustness and redundancy of biological pathways. It has become clear that multifactorial diseases such as cancer, neurological, and inflammatory disorders, may require more complex therapeutic approaches hitting a certain biological system as a whole. Concerning epigenetics, the goal of the multi-epi-target approach consists in the development of small molecules able to simultaneously and (often) reversibly bind different specific epi-targets. To date, two dual histone deacetylase/kinase inhibitors (CUDC-101 and CUDC-907) are in an advanced stage of clinical trials. In the last years, the growing interest in polypharmacology encouraged the publication of high-quality reviews on combination therapy and hybrid molecules. Hence, to update the state-of-the-art of these therapeutic approaches avoiding redundancy, herein we focused only on multiple medication therapies and multitargeting compounds exploiting epigenetic plus nonepigenetic drugs reported in the literature in 2018. In addition, all the multi-epi-target inhibitors known in literature so far, hitting two or more epigenetic targets, have been included.

Identification of Inhibitors to Trypanosoma cruzi Sirtuins Based on Compounds Developed to Human Enzymes.

Chagas disease is an illness caused by the protozoan parasite Trypanosoma cruzi, affecting more than 7 million people in the world. Benznidazole and nifurtimox are the only drugs available for treatment and in addition to causing several side effects, are only satisfactory in the acute phase of the disease. Sirtuins are NAD+-dependent deacetylases involved in several biological processes, which have become drug target candidates in various disease settings. T. cruzi presents two sirtuins, one cytosolic (TcSir2rp1) and the latter mitochondrial (TcSir2rp3). Here, we characterized the effects of human sirtuin inhibitors against T. cruzi sirtuins as an initial approach to develop specific parasite inhibitors. We found that, of 33 compounds tested, two inhibited TcSir2rp1 (15 and 17), while other five inhibited TcSir2rp3 (8, 12, 13, 30, and 32), indicating that specific inhibitors can be devised for each one of the enzymes. Furthermore, all inhibiting compounds prevented parasite proliferation in cultured mammalian cells. When combining the most effective inhibitors with benznidazole at least two compounds, 17 and 32, demonstrated synergistic effects. Altogether, these results support the importance of exploring T. cruzi sirtuins as drug targets and provide key elements to develop specific inhibitors for these enzymes as potential targets for Chagas disease treatment.

Effects of Glutathione Transferase-Targeting Nitrobenzoxadiazole Compounds in Relation to PD-L1 Status in Human Melanoma Cells.

BACKGROUND: PD-L1 is a membrane protein with inhibitory effects on immune responses, whose expression has been correlated with high aggressiveness and the propensity of melanoma to metastasize. The nitrobenzoxadiazole (NBD) NBDHEX and its analog MC3181 are endowed with strong antitumor activity towards melanoma and a significant ability to reduce its adhesion and invasiveness. Therefore, we investigated whether PD-L1 status could affect cell sensitivity to the cytotoxic effects of NBDs. We then evaluated the effects of NBDHEX on PD-L1 expression and autophagy in melanoma cells. We used the BRAF-mutated A375 melanoma cell line and an A375 variant population enriched for PD-L1+ cells as a model. The cytotoxic effects of NBDs were evaluated in comparison to those of the BRAF inhibitor vemurafenib and the autophagy inhibitor chloroquine. METHODS: The effect of NBDHEX on autophagy was determined by measuring LC3-II and p62 protein levels by Western blot. The cytotoxic activity of the compounds was evaluated by sulforhodamine B assay. PD-L1 expression and plasma membrane localization were analyzed by FACS and Western blot analysis. RESULTS: NBDHEX behaves as a late-autophagy inhibitor in A375 melanoma cells, as previously found in other tumor cell lines. NBDHEX and MC3181 showed strong and comparable cytotoxic activity in both parental and PD-L1+ A375 cells, with IC50 values in the sub-micromolar range. Conversely, cells sorted for high PD-L1 expression had lower sensitivity to both the BRAF inhibitor vemurafenib and the autophagy inhibitor chloroquine. NBDHEX treatment did not change the total expression and cell surface localization of PD-L1 in both parental and PD-L1+ A375 cells. CONCLUSIONS: Our data suggest that NBDs may represent a promising treatment strategy for melanoma with elevated PD-L1 expression.

Synthesis and characterisation of a new benzamide-containing nitrobenzoxadiazole as a GSTP1-1 inhibitor endowed with high stability to metabolic hydrolysis.

The antitumor agent 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (1) is a potent inhibitor of GSTP1-1, a glutathione S-transferase capable of inhibiting apoptosis by binding to JNK1 and TRAF2. We recently demonstrated that, unlike its parent compound, the benzoyl ester of 1 (compound 3) exhibits negligible reactivity towards GSH, and has a different mode of interaction with GSTP1-1. Unfortunately, 3 is susceptible to rapid metabolic hydrolysis. In an effort to improve the metabolic stability of 3, its ester group has been replaced by an amide, leading to N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexyl)benzamide (4). Unlike 3, compound 4 was stable to human liver microsomal carboxylesterases, but retained the ability to disrupt the interaction between GSTP1-1 and TRAF2 regardless of GSH levels. Moreover, 4 exhibited both a higher stability in the presence of GSH and a greater cytotoxicity towards cultured A375 melanoma cells, in comparison with 1 and its analog 2. These findings suggest that 4 deserves further preclinical testing.

Enzymatic and Biological Characterization of Novel Sirtuin Modulators against Cancer.

Sirtuins, a family of nicotinamide adenine dinucleotide (NAD+)-dependent lysine deacetylases, are promising targets for anticancer treatment. Recently, we characterized a novel pan-sirtuin (SIRT) inhibitor, MC2494, displaying antiproliferative effects and able to induce death pathways in several human cancer cell lines and decrease tumor growth in vivo. Based on the chemical scaffold of MC2494, and by applying a structure-activity relationship approach, we developed a small library of derivative compounds and extensively analyzed their enzymatic action at cellular level as well as their ability to induce cell death. We also investigated the effect of MC2494 on regulation of cell cycle progression in different cancer cell lines. Our investigations indicated that chemical substitutions applied to MC2494 scaffold did not confer higher efficacy in terms of biological activity and SIRT1 inhibition, but carbethoxy-containing derivatives showed higher SIRT2 specificity. The carbethoxy derivative of MC2494 and its 2-methyl analog displayed the strongest enzymatic activity. Applied chemical modifications improved the enzymatic selectivity of these SIRT inhibitors. Additionally, the observed activity of MC2494 via cell cycle and apoptotic regulation and inhibition of cell migration supports the potential role of SIRTs as targets in tumorigenesis and makes SIRT-targeting molecules good candidates for novel pharmacological approaches in personalized medicine.

H19-Dependent Transcriptional Regulation of ß3 and ß4 Integrins Upon Estrogen and Hypoxia Favors Metastatic Potential in Prostate Cancer.

Estrogen and hypoxia promote an aggressive phenotype in prostate cancer (PCa), driving transcription of progression-associated genes. Here, we molecularly dissect the contribution of long non-coding RNA H19 to PCa metastatic potential under combined stimuli, a topic largely uncovered. The effects of estrogen and hypoxia on H19 and cell adhesion molecules' expression were investigated in PCa cells and PCa-derived organotypic slice cultures (OSCs) by qPCR and Western blot. The molecular mechanism was addressed by chromatin immunoprecipitations, overexpression, and silencing assays. PCa cells' metastatic potential was analyzed by in vitro cell-cell adhesion, motility test, and trans-well invasion assay. We found that combined treatment caused a significant H19 down-regulation as compared with hypoxia. In turn, H19 acts as a transcriptional repressor of cell adhesion molecules, as revealed by up-regulation of both ß3 and ß4 integrins and E-cadherin upon H19 silencing or combined treatment. Importantly, H19 down-regulation and ß integrins induction were also observed in treated OSCs. Combined treatment increased both cell motility and invasion of PCa cells. Lastly, reduction of ß integrins and invasion was achieved through epigenetic modulation of H19-dependent transcription. Our study revealed that estrogen and hypoxia transcriptionally regulate, via H19, cell adhesion molecules redirecting metastatic dissemination from EMT to a ß integrin-mediated invasion.

Disruptor of telomeric silencing 1-like (DOT1L): disclosing a new class of non-nucleoside inhibitors by means of ligand-based and structure-based approaches.

Chemical inhibition of chromatin-mediated signaling involved proteins is an established strategy to drive expression networks and alter disease progression. Protein methyltransferases are among the most studied proteins in epigenetics and, in particular, disruptor of telomeric silencing 1-like (DOT1L) lysine methyltransferase plays a key role in MLL-rearranged acute leukemia Selective inhibition of DOT1L is an established attractive strategy to breakdown aberrant H3K79 methylation and thus overexpression of leukemia genes, and leukemogenesis. Although numerous DOT1L inhibitors have been several structural data published no pronounced computational efforts have been yet reported. In these studies a first tentative of multi-stage and LB/SB combined approach is reported in order to maximize the use of available data. Using co-crystallized ligand/DOT1L complexes, predictive 3-D QSAR and COMBINE models were built through a python implementation of previously reported methodologies. The models, validated by either modeled or experimental external test sets, proved to have good predictive abilities. The application of these models to an internal library led to the selection of two unreported compounds that were found able to inhibit DOT1L at micromolar level. To the best of our knowledge this is the first report of quantitative LB and SB DOT1L inhibitors models and their application to disclose new potential epigenetic modulators.