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.

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.

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.

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.

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.

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.

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.

An in vivo active 1,2,5-oxadiazole Pt(II) complex: A promising anticancer agent endowed with STAT3 inhibitory properties.

New Pt(II) complexes (Pt-1-3) bearing 1,2,5-oxadiazole ligands (1-3) were synthesized, characterized and evaluated for their ability to disrupt STAT3 dimerization. Ligand 3·HCl showed cytotoxic effects on HCT-116 cells (IC50 = 95.2 µM) and a selective ability to interact with STAT3 (IC50 = 8.2 µM) versus STAT1 (IC50 > 30 µM). Its corresponding platinum complex Pt-3 exhibited an increased cytotoxicity (IC50 = 18.4 µM) and a stronger interaction with STAT3 (IC50 = 1.4 µM), leading to inhibition of its signaling pathway. Pt-3 was also evaluated in cell-based assays for its action on p53 expression and on STAT3 phosphorylation. In syngeneic murine Lewis lung carcinoma (LLC) implanted in C57BL/6 mice, Pt-3 showed a higher antitumor activity with fewer side effects than cisplatin.

Iron Acquisition Pathways as Targets for Antitubercular Drugs.

Tuberculosis nowadays ranks as the second leading cause of death from an infectious disease worldwide. In the last twenty years, this disease has again started to spread mainly for the appearance of multi-drug resistant forms. Therefore, new targets are needed to address the growing emergence of bacterial resistance and for antitubercular drug development. Efficient iron acquisition is crucial for the pathogenesis of Mycobacterium tuberculosis, because it serves as cofactor in many essential biological processes, including DNA biosynthesis and cellular respiration. Bacteria acquire iron chelating non-heme iron from the host using the siderophore mycobactins and carboxymycobactins and by the uptake of heme iron released by damaged red blood cells, through several acquisition systems. Drug discovery focused its efforts on the inhibition of MbtI and MbtA, which are are two enzymes involved in the mycobactin biosynthetic pathway. In particular, MbtI inhibitors have been studied in vitro, while MbtA inhibitors showed activity also in infected mice. Another class of compounds, MmpL3 inhibitors, showed antitubercular activity in vitro and in vivo, but their mechanism of action seems to be off-target. Some compounds inhibiting 4'-phosphopantetheinyl transferase were discovered but not tested on in vivo assays. The available data reported in this study based on inhibitors and gene deletion studies, suggest that targeting iron acquisition systems could be considered a promising antitubercular strategy. Due to their redundancy, the relative importance of each pathway for Mycobacterium tuberculosis survival has still to be determined. Thus, in vivo studies with new, potent and specific inhibitors are needed to highlight target selection.

Identification of a new STAT3 dimerization inhibitor through a pharmacophore-based virtual screening approach.

Signal transducer and activator of transcription 3 (STAT3) plays an essential role in cell growth regulation and survival. An aberrant STAT3 activation and/or expression is implied in various solid and blood tumors as well as in other pathologies like rheumatoid arthritis and pulmonary fibrosis, thus making the search for STAT3 inhibitors a growing field of study. With the aim of identifying new inhibitors of STAT3 dimerization, we screened a database including more than 1 320 000 commercially available compounds using a receptor-based pharmacophore model comprising the key protein-protein interactions identified in the STAT3 dimer and refining the search through docking and molecular dynamic simulations studies. STAT3 binding assays revealed a significant STAT3 inhibitory activity and selectivity versus Grb2 for one of the four top-scored compounds, thus verifying the reliability of the virtual screening workflow. Moreover, such compound could already be considered as a lead for the development of new and more potent STAT3 dimerization inhibitors.

The influence of the substitution pattern on the molecular conformation of ureido-1,2,5-oxadiazoles, related to STAT3 inhibitors: chemical behavior and structural investigation.

Signal transducer and activator of transcription 3 (STAT3) is a protein constitutively activated by aberrant upstream tyrosine kinase activities in a broad spectrum of human solid and blood tumors. Therefore, the availability of drugs affecting STAT3 may have important therapeutic potential for the treatment of cancer. Pursuing our efforts in exploring the influence of the substitution pattern of the ureido 1,2,5-oxadiazole moiety on the molecular conformation, new compounds substituted at positions 3 and 4 on the furazane ring were synthesized. The inhibition properties vs. STAT3 of the novel compounds were evaluated in a dual-luciferase assay, using HCT-116 cells, and the results evidenced a moderate activity only for the compounds endowed with a planar arrangement. Crystallographic studies of the new derivatives were performed in order to evidence the peculiar chemical behavior and to evaluate how structural modulations affected the biological properties.

Signal transducer and activator of transcription 3 (STAT3): a promising target for anticancer therapy.

Signal transducer and activator of transcription 3 (STAT3) is an oncogenic protein whose inhibition is sought for the prevention and treatment of cancer. In this review, the validated therapeutic strategy to block aberrant activity of STAT3 in many tumor cell lines is evaluated by presenting the most promising inhibitors to date. The compounds are discussed in classes based on their different mechanisms of action, which are critically explained. In addition, their future clinical development as anticancer agents is considered. Furthermore, the efforts devoted to the comprehension of the structure-activity relationships and to the identification of the biological effects are brought to attention. The synthetic and technological approaches recently developed to overcome the difficulties in the obtainment of clinically suitable drugs are also presented.

ortho-Halogen naphthaleins as specific inhibitors of Lactobacillus casei thymidylate synthase. Conformational properties and biological activity.

Thymidylate synthase (TS) (EC 2.1.1.45), an enzyme involved in the DNA synthesis of both prokaryotic and eukaryotic cells, is a potential target for the development of anticancer and antinfective agents. Recently, we described a series of phthalein and naphthalein derivatives as TS inhibitors. These compounds have structures unrelated to the folate (Non-Analogue Antifolate Inhibitors, NAAIs) and were selective for the bacterial versus the human TS (hTS). In particular, halogen-substituted molecules were the most interesting. In the present paper the halogen derivatives of variously substituted 3,3-bis(4-hydroxyphenyl)-1H,3H-naphtho[2,3-c]furan-1-one (1-5) and 3,3-bis(4-hydroxyphenyl)-1H,3H-naphtho[1,8-c,d]pyran-1-one (6-14) were synthesized to investigate the biological effect of halogen substitution on the inhibition and selectivity for the TS enzymes. Conformational properties of the naphthalein series were explored in order to highlight possible differences between molecules that show species-specific biological profile with respect to non species-specific ones. With this aim, the conformational properties of the synthesized compounds were investigated by NMR, in various solvents and at different temperatures, and by computational analysis. The apparent inhibition constants (K(i)) for Lactobacillus casei TS (LcTS) were found to range from 0.7 to 7.0 microM, with the exception of the weakly active iodo-derivatives (4, 10, 13); all] the compounds were poorly active against hTS. The di-halogenated compounds 7, 8, 14 showed the highest specificity towards LcTS, their specificity index (SI) ranging between 40 and >558. The di-halogenated 1,8-naphthalein derivatives (7-10) exhibited different conformational properties with respect to the tetra-haloderivatives. Though a clear explanation for the observed specificity by means of conformational analysis is difficult to find, some interesting conformational effects are discussed in the context of selective recognition of the compounds investigated by the LcTS enzyme.