Structural basis for specific inhibition of salicylate synthase from Mycobacterium abscessus.

Blocking iron uptake and metabolism has been emerging as a promising therapeutic strategy for the development of novel antimicrobial compounds. Like all mycobacteria, M. abscessus (Mab) has evolved several countermeasures to scavenge iron from host carrier proteins, including the production of siderophores, which play a crucial role in these processes. In this study, we solved, for the first time, the crystal structure of Mab-SaS, the first enzyme involved in the biosynthesis of siderophores. Moreover, we screened a small, focused library and identified a compound exhibiting a potent inhibitory effect against Mab-SaS (IC50 ≈ 2 µM). Its binding mode was investigated by means of Induced Fit Docking simulations, performed on the crystal structure presented herein. Furthermore, cytotoxicity data and pharmacokinetic predictions revealed the safety and drug-likeness of this class of compounds. Finally, the crystallographic data were used to optimize the model for future virtual screening campaigns. Taken together, the findings of our study pave the way for the identification of potent Mab-SaS inhibitors, based on both established and unexplored chemotypes.

Eco-Friendly Bio-Based Solvents for the Acetylation of the Amino Group of Amino Acids.

Nature-derived products, like juices and peel extracts of fruits and vegetables, have emerged in recent years as interesting and sustainable alternatives to traditional solvents in several synthetic applications. Herein, we present a green and fast method for the N-acetylation of amino acids, using several bio-based solvents (vinegar, tomato/kiwi/apple peel extracts, lemon juice, etc.). The high reactivity of the amino group is often a limitation in synthetic processes, making its protection a necessary step to achieve pure products and limit side reactions. Therefore, versatile, time-efficient procedures, minimal purification efforts, and good yields are desirable features for these transformations. Our new method meets all these criteria, offering a valuable and eco-friendly alternative to traditional approaches. In detail, we managed to obtain comparable yields to established setups, while improving safety and reducing the environmental impact of the overall process. Most notably, the milder conditions made it possible to avoid the use of running water (saving about 250 L/reaction) and electric-powered cooling devices.

Targeting Siderophore-Mediated Iron Uptake in M. abscessus: A New Strategy to Limit the Virulence of Non-Tuberculous Mycobacteria.

Targeting pathogenic mechanisms, rather than essential processes, represents a very attractive approach for the development of new antimycobacterial drugs. In this context, iron acquisition routes have recently emerged as potentially druggable pathways. However, the importance of siderophore biosynthesis in the virulence and pathogenicity of M. abscessus (Mab) is still poorly understood. In this study, we investigated the Salicylate Synthase (SaS) of Mab as an innovative molecular target for the development of inhibitors of siderophore production. Notably, Mab-SaS does not have any counterpart in human cells, making it an interesting candidate for drug discovery. Starting from the analysis of the binding of a series of furan-based derivatives, previously identified by our group as inhibitors of MbtI from M. tuberculosis (Mtb), we successfully selected the lead compound 1, exhibiting a strong activity against Mab-SaS (IC50 ≈ 5 µM). Computational studies characterized the key interactions between 1 and the enzyme, highlighting the important roles of Y387, G421, and K207, the latter being one of the residues involved in the first step of the catalytic reaction. These results support the hypothesis that 5-phenylfuran-2-carboxylic acids are also a promising class of Mab-SaS inhibitors, paving the way for the optimization and rational design of more potent derivatives.

Synthesis and Assessment of the In Vitro and Ex Vivo Activity of Salicylate Synthase (Mbti) Inhibitors as New Candidates for the Treatment of Mycobacterial Infections.

Tuberculosis (TB) causes millions of deaths every year, ranking as one of the most dangerous infectious diseases worldwide. Because several pathogenic strains of M. tuberculosis (Mtb) have developed resistance against most of the established anti-TB drugs, new therapeutic options are urgently needed. An attractive target for the development of new anti-TB agents is the salicylate synthase MbtI, the first enzyme of the mycobacterial siderophore biochemical machinery, absent in human cells. In this work, a set of analogues of 5-(3-cyanophenyl)furan-2-carboxylic acid (I), the most potent MbtI inhibitor identified to date, was synthesized, characterized, and tested to further elucidate the structural requirements for achieving an efficient MbtI inhibition and potent antitubercular activity. The structure-activity relationships (SAR) discussed herein evidenced the importance of the side chain linked to the phenyl moiety to improve the in vitro antimycobacterial activity. In detail, 1f emerged as the most effective analogue against the pathogen, acting without cytotoxicity issues. To deepen the understanding of its mechanism of action, we established a fluorescence-based screening test to quantify the pathogen infectivity within host cells, using MPI-2 murine cells, a robust surrogate for alveolar macrophages. The set-up of the new assay demonstrates significant potential to accelerate the discovery of new anti-TB drugs.

Insights on the Modulation of SIRT5 Activity: A Challenging Balance.

SIRT5 is a member of the Sirtuin family, a class of deacetylating enzymes consisting of seven isoforms, involved in the regulation of several processes, including gene expression, metabolism, stress response, and aging. Considering that the anomalous activity of SIRT5 is linked to many pathological conditions, we present herein an overview of the most interesting modulators, with the aim of contributing to further development in this field.

Natural products against key Mycobacterium tuberculosis enzymatic targets: Emerging opportunities for drug discovery.

For centuries, natural products (NPs) have served as powerful therapeutics against a variety of human ailments. Nowadays, they still represent invaluable resources for the treatment of many diseases, including bacterial infections. After nearly three decades since the World Health Organization's (WHO) declaration of tuberculosis (TB) as a global health emergency, Mycobacterium tuberculosis (Mtb) continues to claim millions of lives, remaining among the leading causes of death worldwide. In the last years, several efforts have been devoted to shortening and improving treatment outcomes, and to overcoming the increasing resistance phenomenon. Nature has always provided a virtually unlimited source of bioactive molecules, which have inspired the development of new drugs. NPs are characterized by an exceptional chemical and structural diversity, the result of millennia of evolutionary responses to various stimuli. Thanks to their favorable structural features and their enzymatic origin, they are naturally prone to bind proteins and exhibit bioactivities. Furthermore, their worldwide distribution and ease of accessibility has contributed to promote investigations on their activity. Overall, these characteristics make NPs excellent models for the design of novel therapeutics. This review offers a critical and comprehensive overview of the most promising NPs, isolated from plants, fungi, marine species, and bacteria, endowed with inhibitory properties against traditional and emerging mycobacterial enzymatic targets. A selection of 86 compounds is here discussed, with a special emphasis on their biological activity, structure-activity relationships, and mechanism of action. Our study corroborates the antimycobacterial potential of NPs, substantiating their relevance in future drug discovery and development efforts.

Synthesis, Characterization, and Biological Evaluation of New Derivatives Targeting MbtI as Antitubercular Agents.

Tuberculosis (TB) causes millions of deaths every year, ranking as one of the most dangerous infectious diseases worldwide. Because several pathogenic strains of Mycobacterium tuberculosis (Mtb) have developed resistance against most of the established anti-TB drugs, new therapeutic options are urgently needed. An attractive target for the development of new antitubercular agents is the salicylate synthase MbtI, an essential enzyme for the mycobacterial siderophore biochemical machinery, absent in human cells. A set of analogues of I and II, two of the most potent MbtI inhibitors identified to date, was synthesized, characterized, and tested to elucidate the structural requirements for achieving an efficient MbtI inhibition and a potent antitubercular activity with this class of compounds. The structure-activity relationships (SAR) here discussed evidenced the importance of the furan as part of the pharmacophore and led to the preparation of six new compounds (IV-IX), which gave us the opportunity to examine a hitherto unexplored position of the phenyl ring. Among them emerged 5-(3-cyano-5-(trifluoromethyl)phenyl)furan-2-carboxylic acid (IV), endowed with comparable inhibitory properties to the previous leads, but a better antitubercular activity, which is a key issue in MbtI inhibitor research. Therefore, compound IV offers promising prospects for future studies on the development of novel agents against mycobacterial infections.

Towards the Inhibition of Protein-Protein Interactions (PPIs) in STAT3: Insights into a New Class of Benzothiadiazole Derivatives.

Signal transducer and activator of transcription 3 (STAT3) is a validated anticancer target due to the relationship between its constitutive activation and malignant tumors. Through a virtual screening approach on the STAT3-SH2 domain, 5,6-dimethyl-1H,3H-2,1,3-benzothiadiazole-2,2-dioxide (1) was identified as a potential STAT3 inhibitor. Some benzothiadiazole derivatives were synthesized by employing a versatile methodology, and they were tested by an AlphaScreen-based assay. Among them, benzosulfamide 1 showed a significant activity with an IC50 = 15.8 ± 0.6 µM as a direct STAT3 inhibitor. Notably, we discovered that compound 1 was also able to interact with cysteine residues located around the SH2 domain. By applying mass spectrometry, liquid chromatography, NMR, and UV spectroscopy, an in-depth investigation was carried out, shedding light on its intriguing and unexpected mechanism of interaction.

Shedding X-ray Light on the Role of Magnesium in the Activity of Mycobacterium tuberculosis Salicylate Synthase (MbtI) for Drug Design.

The Mg2+-dependent Mycobacterium tuberculosis salicylate synthase (MbtI) is a key enzyme involved in the biosynthesis of siderophores. Because iron is essential for the survival and pathogenicity of the microorganism, this protein constitutes an attractive target for antitubercular therapy, also considering the absence of homologous enzymes in mammals. An extension of the structure-activity relationships of our furan-based candidates allowed us to disclose the most potent competitive inhibitor known to date (10, Ki = 4 µM), which also proved effective on mycobacterial cultures. By structural studies, we characterized its unexpected Mg2+-independent binding mode. We also investigated the role of the Mg2+ cofactor in catalysis, analyzing the first crystal structure of the MbtI-Mg2+-salicylate ternary complex. Overall, these results pave the way for the development of novel antituberculars through the rational design of improved MbtI inhibitors.

Discovery of Novel Naphthylphenylketone and Naphthylphenylamine Derivatives as Cell Division Cycle 25B (CDC25B) Phosphatase Inhibitors: Design, Synthesis, Inhibition Mechanism, and in Vitro Efficacy against Melanoma Cell Lines.

CDC25 phosphatases play a critical role in the regulation of the cell cycle and thus represent attractive cancer therapeutic targets. We previously discovered the 4-(2-carboxybenzoyl)phthalic acid (NSC28620) as a new CDC25 inhibitor endowed with promising anticancer activity in breast, prostate, and leukemia cells. Herein, we report a structure-based optimization of NSC28620, leading to the identification of a series of novel naphthylphenylketone and naphthylphenylamine derivatives as CDC25B inhibitors. Compounds 7j, 7i, 6e, 7f, and 3 showed higher inhibitory activity than the initial lead, with Ki values in the low micromolar range. Kinetic analysis, intrinsic fluorescence studies, and induced fit docking simulations provided a mechanistic understanding of the activity of these derivatives. All compounds were tested in the highly aggressive human melanoma cell lines A2058 and A375. Compound 4a potently inhibited cell proliferation and colony formation, causing an increase of the G2/M phase and a reduction of the G0/G1 phase of the cell cycle in both cell lines.

Unravelling the Antiproliferative Activity of 1,2,5-oxadiazole Derivatives.

AIM: To develop several new derivatives aimed to complete the studies concerning the antiproliferative profile of the oxadiazole derivative MD77. MATERIALS AND METHODS: The substitution pattern around the phenyl rings of this compound was analyzed through the synthesis of positional isomers and of analogues bearing different substituents at the para positions (2-12). RESULTS: The results of the antiproliferative activity of these derivatives versus HCT-116 and HeLa cancer cell lines shed light on the effects of the presence, nature and position of such substituents. Notably, derivative 4, a regioisomer of 1 in which the substituents at the para positions of the phenyl rings were inverted, showed the best antiproliferative profile, exhibiting a significant activity also against MCF7 and MDA-MB 468 cancer cell lines. CONCLUSION: Preliminary results showed the ability of compound 4 to reduce the viability of cancer cells by counteracting human recombinant topoisomerase II α relaxation activity.

An Overview on the Potential Antimycobacterial Agents Targeting Serine/Threonine Protein Kinases from Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), still remains an urgent global health issue, mainly due to the emergence of multi-drug resistant strains. Therefore, there is a pressing need to develop novel and more efficient drugs to control the disease. In this context, targeting the pathogen virulence factors, and particularly signal mechanisms, seems to be a promising approach. An important transmembrane signaling system in Mtb is represented by receptor-type Serine/ Threonine protein kinases (STPKs). Mtb has 11 different STPKs, two of them, PknA and PknB, are essential. By contrast PknG and PknH are involved in Mtb virulence and adaptation, and are fundamental for the pathogen growth in infection models. Therefore, STPKs represent a very interesting group of pharmacological targets in M. tuberculosis. In this work, the principal inhibitors of the mycobacterial STPKs will be presented and discussed. In particular, medicinal chemistry efforts have been focused on discovering new antimycobacterial compounds, targeting three of these kinases, namely PknA, PknB and PknG. Generally, the inhibitory effect on these enzymes do not correlate with a significant antimycobacterial action in whole-cell assays. However, compounds with activity in the low micromolar range have been obtained, demonstrating that targeting Mtb STPKs could be a new promising strategy for the development of drugs to treat TB infections.

New insight into structure-activity of furan-based salicylate synthase (MbtI) inhibitors as potential antitubercular agents.

Starting from the analysis of the hypothetical binding mode of our previous furan-based hit (I), we successfully achieved our objective to replace the nitro moiety, leading to the disclosure of a new lead exhibiting a strong activity against MbtI. Our best candidate 1 h displayed a Ki of 8.8 µM and its antimycobacterial activity (MIC99 = 250 µM) is conceivably related to mycobactin biosynthesis inhibition. These results support the hypothesis that 5-phenylfuran-2-carboxylic derivatives are a promising class of MbtI inhibitors.

Exploring the Biological Activity of a Library of 1,2,5-Oxadiazole Derivatives Endowed With Antiproliferative Activity.

BACKGROUND/AIM: The identification of a series of oxadiazole-based compounds, as promising antiproliferative agents, has been previously reported. The aim of this study was to explore the SAR of newly-synthesized oxadiazole derivatives and identify their molecular targets. MATERIALS AND METHODS: A small library of 1,2,5-oxadiazole derivatives was synthetized and their antiproliferative activity was tested by the MTT assay. Their interaction with topoisomerase I was evaluated and a molecular docking study was performed. RESULTS: Several candidates showed cytotoxicity towards two human tumor cell lines, HCT-116 (colorectal carcinoma) and HeLa (cervix adenocarcinoma). Some derivatives exhibited inhibitory effects on the catalytic activity of topoisomerase I and this effect was supported by docking studies. CONCLUSION: The enzyme inhibition results, although not directly related to cytotoxicity, suggest that a properly modified 1,2,5 oxadiazole scaffold could be considered for the development of new anti-topoisomerase agents.

Signal Transducer and Activator of Transcription Protein 3 (STAT3): An Update on its Direct Inhibitors as Promising Anticancer Agents.

BACKGROUND: Since Signal Transducer and Activator of Transcription 3 (STAT3) is a transcription factor which plays an important role in multiple aspects of cancer, including progression and migration, and it is constitutively activated in various human tumors, STAT3 inhibition has emerged as a validated strategy for the treatment of several malignancies. The aim of this review is to provide an update on the identification of new promising direct inhibitors targeting STAT3 domains, as potential anticancer agents. METHODS: A thorough literature search focused on recently reported STAT3 direct inhibitors was undertaken. We considered the relevant developments regarding the STAT3 domains, which have been identified as potential drug targets. RESULTS: In detail, 135 peer-reviewed papers and 7 patents were cited; the inhibitors we took into account targeted the DNA binding domain (compounds were grouped into natural derivatives, small molecules, peptides, aptamers and oligonucleotides), the SH2 binding domain (natural, semi-synthetic and synthetic compounds) and specific residues, like cysteines (natural, semi-synthetic, synthetic compounds and dual inhibitors) and tyrosine 705. CONCLUSION: The huge number of direct STAT3 inhibitors recently identified demonstrates a strong interest in the investigation of this target, although it represents a challenging task considering that no drug targeting this enzyme is currently available for anticancer therapy. Notably, many studies on the available inhibitors evidenced that some of them possess a dual mechanism of action.

Discovery and development of novel salicylate synthase (MbtI) furanic inhibitors as antitubercular agents.

We report on the virtual screening, synthesis, and biological evaluation of new furan derivatives targeting Mycobacterium tuberculosis salicylate synthase (MbtI). A receptor-based virtual screening procedure was applied to screen the Enamine database, identifying two compounds, I and III, endowed with a good enzyme inhibitory activity. Considering the most active compound I as starting point for the development of novel MbtI inhibitors, we obtained new derivatives based on the furan scaffold. Among the SAR performed on this class, compound 1a emerged as the most potent MbtI inhibitor reported to date (Ki = 5.3 µM). Moreover, compound 1a showed a promising antimycobacterial activity (MIC99 = 156 µM), which is conceivably related to mycobactin biosynthesis inhibition.

New Chromane-Based Derivatives as Inhibitors of Mycobacterium tuberculosis Salicylate Synthase (MbtI): Preliminary Biological Evaluation and Molecular Modeling Studies.

Tuberculosis is the leading cause of death from a single infectious agent worldwide; therefore, the need for new antitubercular drugs is desperate. The recently validated target salicylate synthase MbtI is the first enzyme involved in the biosynthesis of mycobactins, compounds able to chelate iron, an essential cofactor for the survival of Mycobacterium tuberculosis in the host. Here, we report on the synthesis and biological evaluation of chromane-based compounds as new potential inhibitors of MbtI. Our approach successfully allowed the identification of a novel lead compound (1), endowed with a promising activity against this enzyme (IC50 = 55 µM). Molecular modeling studies were performed in order to evaluate the binding mode of 1 and rationalize the preliminary structure-activity relationships, thus providing crucial information to carry out further optimization studies.

Structure-based drug design, synthesis and biological assays of P. falciparum Atg3-Atg8 protein-protein interaction inhibitors.

The proteins involved in the autophagy (Atg) pathway have recently been considered promising targets for the development of new antimalarial drugs. In particular, inhibitors of the protein-protein interaction (PPI) between Atg3 and Atg8 of Plasmodium falciparum retarded the blood- and liver-stages of parasite growth. In this paper, we used computational techniques to design a new class of peptidomimetics mimicking the Atg3 interaction motif, which were then synthesized by click-chemistry. Surface plasmon resonance has been employed to measure the ability of these compounds to inhibit the Atg3-Atg8 reciprocal protein-protein interaction. Moreover, P. falciparum growth inhibition in red blood cell cultures was evaluated as well as the cyto-toxicity of the compounds.

Low density polyethylene functionalized with antibiofilm compounds inhibits Escherichia coli cell adhesion.

The present work concerns an efficient strategy to obtain novel medical devices materials able to inhibit biofilm formation. The new materials were achieved by covalent grafting of p-aminocinnamic or p-aminosalicylic acids on low density polyethylene coupons. The polyethylene surface, previously activated by oxygen plasma treatment, was functionalized using 2-hydroxymethylmetacrylate as linker. The latter was reacted with succinic anhydride affording the carboxylic end useful for the immobilization of the antibiofilm molecules. The modified surface was characterized by scanning electron microscope, X-ray photoelectron spectroscopy, attenuated total reflectance Fourier transform infrared and fluorescence analyses. The antibiofilm activity of the modified materials were tested against Escherichia coli biofilm grown in the Center of Disease Control biofilm reactor. The results revealed that the grafted cinnamic and salicylic acid derivatives reduced biofilm biomass, in comparison with the control, by 73.7 ± 10.7% and 63.4 ± 7.1%, respectively. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3251-3261, 2017.

A field-based disparity analysis of new 1,2,5-oxadiazole derivatives endowed with antiproliferative activity.

A series of 1,2,5-oxadiazoles were synthesized as new potential antiproliferative agents. The in vitro cytotoxic activity evaluation of title compounds through MTT assay revealed that some of them showed significant activity against the HCT-116 cancer cell line. The field-based disparity analysis provided indications about the electrostatic, hydrophobic, and shape features underlying the cytotoxicity, suggesting that increasing the negative electrostatic field on the heterocyclic core of the structure has positive effects on the activity. The structure-activity relationships (SAR) around a particular compound can be explained allowing for a structural rationale for the differences in activity. The SAR provided by this series of compounds can be exploited to carry out further lead optimization.