25-Hydroxycholesterol (25-HC) and 25R,26-hydroxycholesterol (25R,26-HC) are two endogenous oxysterols endowed with broad-spectrum antiviral activity. They hamper viral replication by targeting oxysterol-binding protein (OSBP), a host lipid transporter which plays a critical role in the replicative cycle of several viruses. Recently, we have reported the remarkable antiviral activity against herpes simplex virus 2 (HSV-2) of N,N-dimethyl-3β-hydroxychol-5-en-24-amide (PFM067, 3), identified by the screening of our synthetic oxysterol library. Further development of antiviral cholenamide-based fluorescent probes allowed us to support the hypothesis that OSBP was involved in the antiviral mechanism of action of the parent PFM067. Herein, we report the first study aimed at the definition of structure-antiviral activity relationships for PFM067 (3); successfully, we identified 24-(morpholin-4-yl)-3β-hydroxychol-5-en-24-one (8d), as highly potent, nanomolar inhibitor of HSV-2 replication, endowed with promising selectivity index. Moreover, we demonstrated that OSBP was the molecular target underlying the anti-HSV-2 activity of PFM067 (3) and its analogues by the development of a microscale thermophoresis binding assay. In-depth experiments performed using an OSBP-silenced cell line allowed us to disclose the role played by OSBP in the replicative cycle of HSV-2. Finally, molecular modelling studies evidenced a peculiar orientation of the active compound 8d within the binding site of OSBP respect to that stroked by either the endogenous ligand or an inactive compound.

ABSTRACT:Despite great progress in understanding the genomic basis of immature T-cell acute lymphoblastic leukemia/lymphoblastic lymphoma (T-ALL) and acute leukemias of ambiguous lineage, there are still cases that lack defining genetic markers, complicating risk stratification, and limiting targeted therapeutic options. Recent studies have shown that enhancer hijacking drives oncogene activation in approximately half of T-ALL cases, with the BCL11B (BCL11 transcription factor B) enhancer frequently involved. Here, we describe a subtype of leukemia with a distinct gene-expression signature, and immunophenotype characterized by positivity for immature (CD38), myeloid (CD13), T-lymphoid (cytoplasmic [c]CD3, CD7), and B-lymphoid markers (CD19, CD79a, CD10). This subtype is defined by the t(14;16)(q32;q24) translocation, which places the FOXF1 gene and its antisense long noncoding RNA gene FENDRR under the regulatory control of the BCL11B enhancer, leading to their ectopic transcriptional activation. Common concomitant genetic lesions are loss-of-function alterations of GATA3, CDKN2A/CDKN2B deletion and activating JAK/STAT and NOTCH1 pathway mutations. Patients were predominantly children and adolescents/young adults and experienced poor treatment outcome. High-throughput drug screening of 176 compounds demonstrated efficacy of combined BCL2 apoptosis regulator family proteins and JAK/STAT signaling inhibitors. Additionally, the clinical use of tyrosine kinase inhibitors in some of these patients showed therapeutic efficacy. Collectively, these findings identify BCL11B-enhancer-mediated deregulation of FOXF1/FENDRR as a hallmark of a subtype of high-risk lineage ambiguous leukemia that is potentially amenable to targeted therapeutic intervention.

Nitric oxide (NO) is a central signaling molecule that regulates vascular tone, neurotransmission, and immune function. NO production from L-arginine by nitric oxide synthase (NOS) must be tightly controlled, as excess NO contributes to oxidative and nitrosative stress. Asymmetric dimethylarginine (ADMA) and monomethylarginine (L-NMMA) are endogenous NOS inhibitors whose accumulation leads to cardiovascular, renal, and neurodegenerative diseases. Dimethylarginine dimethylaminohydrolase (DDAH) converts ADMA and NMMA to L-citrulline and dimethylamine or monomethylamine, respectively, thereby controlling NO bioavailability. Two DDAH isoforms are present in humans, namely, DDAH1 and DDAH2. DDAH1 has a demonstrated role in the catabolism of ADMA, while DDAH2 appears to be functionally divergent, playing potential regulatory and signaling roles. Structural analyses reveal that, despite a conserved α/β-propeller architecture, the different biochemical properties of the two isoforms depend on key amino acid substitutions within the active site. Dysregulation of DDAH1 leads to pathological ADMA accumulation, linking impaired enzyme function to endothelial dysfunction and oxidative stress. Consequently, DDAH1 represents an emerging therapeutic target, and several substrate analogues, small molecules, and natural ligands have been identified as inhibitors. On the other hand, the assessment of DDAH2 function is still to be defined. The present review summarizes current molecular, biochemical, and structural insights into DDAH isoforms and discusses their potential as pharmacological targets for diseases associated with altered NO signaling.

A plethora of studies have demonstrated the pathophysiological roles played by paraxonase 2 (PON2) in oxidative stress control, inhibition of apoptosis, infections, and the progression of various types of malignancies. The continuous interest in PON2 has not gone hand in hand with the development of its inhibitors. Indeed, only one inhibitor for PON2, namely TQ416, is known, although neither its preparation nor a systematic structure-activity relationship analysis has been so far reported. Herein, we outline the first study aimed at the definition of structure-activity relationships of TQ416 by the preparation of a small library of its analogues. Successfully, we identified some [1,2,4]triazolo[4,3-a]quinoline derivatives more potent than TQ416 as PON2 inhibitors, and among them one endowed with an IC50 value in the nanomolar range. We tested the parent TQ416 and its most effective congener 2 in cells showing their effectiveness and complex behaviour.

Given the strict regulations governing the introduction of new drugs, chirality and related issues have garnered increasing attention in both modern pharmaceutical industries and academic research settings worldwide, especially in the realm of medicinal chemistry projects. Considering the crucial chirality-related issues, the necessity to establish suitable methodologies for determining and monitoring enantiomeric composition in either medicinal products or drug candidates becomes apparent. Among these methods, those utilizing the high-performance liquid chromatography (HPLC) technique through the "direct approach" with chiral stationary phase (CSP) currently offer distinct advantages over the others due to their efficiency and reliability. Hundreds of CSPs are now available on the market, with around 20-30 CSPs, or even fewer, being most commonly utilized to address the majority of enantiomer separation challenges across various compound classes. In this chapter, six separate examples concerning the enantioseparation of medicinal chemistry-relevant compounds of varying chemical and physicochemical nature are presented. In this framework, the use of both high-molecular-weight (polysaccharide- and glycopeptide-based) and low-molecular-weight (amino acid-, Cinchona alkaloid- and fully synthetic-based) chiral selectors is described under the most relevant elution regimens for liquid chromatography (i.e., reversed-phase (RP), normal-phase (NP), and polar organic (PO)/polar ionic (PI)modes).

Indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors have been developed with the aim of reinvigorating antitumor T-cell responses in the tumor microenvironment by blocking the conversion of the essential amino acid tryptophan into immunoregulatory kynurenines. The lack of efficacy demonstrated in the clinical trials prompts us to revise the “on-target” mechanism of these molecules. By studying the turnover of IDO1 protein in human tumor cells exposed to various IDO1 catalytic inhibitors, such as epacadostat, linrodostat, and navoximod, we show here that these molecules stabilize a non-enzymatic protein conformation of IDO1, independently of their mechanism of inhibition. In the thyroid carcinoma cell line FTC-133, the stabilized and non-enzymatic IDO1 protein promotes the proliferation and migration of the tumor, resulting in an adverse pro-tumorigenic effect. These results uncover an unexpected adverse effect of IDO1 inhibitors in the tumor microenvironment that overcomes the enzymatic inhibition of IDO1, and suggest protein degradation, rather than enzymatic inhibition, as a more effective approach to target IDO1 in the tumor microenvironment.

IntroductionIn the era of targeted therapies, molecules for the reactivation of the oncosuppressor p53 in human cancer have not yet reached FDA or EMA approval. Recently, the interaction region of the MDM2/MDM4 heterodimer, the most efficient inhibitor of p53 levels and function, has been successfully targeted. Disruption of the heterodimer activated p53 oncosuppressive function in vitro and in vivo. Despite these encouraging results, further studies on the MDM2/MDM4 interaction region have yet to progress.MethodsHere, we undertook a detailed bioinformatic and biochemical analysis of this region. Using molecular dynamics simulation followed by umbrella sampling.Results and discussionWe characterized a short peptide and modified derivatives with increased binding affinity and pharmacodynamics features compared to previous molecules. Our results uncover the intrinsic plasticity of the MDM2 RING domain through different binding clefts and provide evidence of its ability to host different peptides by key residues. This data may guide the development of next-generation therapeutic inhibitors.

Owing to the importance of tracing new routes in the development of macromolecular prodrugs, in the present work, two potential macromolecular ester prodrugs (i.e., 12a and 13a ) of selective cyclooxygenase‐2 (COX‐2) inhibitor 7b are designed and synthesized. In the design, two different oligo(ethylene glycol)‐based spacers are linked through a ferulate residue to the backbone hyaluronic acid ( HA ) showing a medium molar mass value (i.e., Mw = 270 kDa). The spacers are designed to differ in the sensitivity to the hydrolytic conditions so that the chemical hydrolysis of ferulate ester bond in 12a is assumed to produce the corresponding ferulic acid derivative 12b . On the other hand, the same reaction in 13a leading to ferulate derivative 13b could be accompanied by the hydrolysis of the second ester bond with the release of the selective COX‐2 inhibitor 7b . The COX inhibitory activity of the newly synthesized compounds is evaluated in vitro, and macromolecular ester prodrugs 12a and 13a are found to be completely inactive together with hydrolysis product 12b . Conversely, these in vitro studies reveal the intriguing COX‐2 inhibitory activity and selectivity of ferulate derivative 13b related to macromolecular ester prodrug 13a . Therefore, to obtain information on the hydrolysis process in different environments, hydrolysis studies are performed on macromolecular ester prodrug 13a by using 1 H NMR and UHPLC‐MS techniques. These studies show that severe hydrolytic conditions (i.e., aqueous NaOH solutions) promote the rapid release of potent and selective COX‐2 inhibitor 7b , whereas in ammonium acetate buffer the release is slower. Overall, these results lead to envision possible applications of the design approach to the development of macromolecular ester prodrugs of all the drug molecules bearing hydroxyl groups in their structures.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a moonlight protein endowed with catalytic and signaling functions. It plays a pivotal role in the immune breaking mechanism leading to immunosuppression in cancer microenvironment. Intense research efforts have been devoted to designing catalytic inhibitors for developing immunotherapies, yet neglecting the enzyme’s signaling function. A few IDO1 inhibitors have reached the clinical stage, including navoximod, epacadostat and linrodostat. Using second harmonic generation analysis (SHG) and molecular dynamics simulations, here we show that these clinical inhibitors can induce distinct allosteric motions in the enzyme that affect the stability of the transient signaling complex between IDO1 and Src tyrosine kinase. Next generation sequencing demonstrates that, despite sharing a similar ability to inhibit the enzyme’s catalytic function, all three catalytic inhibitors modulate the IDO1’s signaling function in different ways, regulating distinct transcriptomes in SKOV3 cells. Indoleamine 2,3-dioxygenase 1 (IDO1) is crucial in cancer-related immunosuppression, yet its signaling function remains underexplored in immunotherapy development. Here, the authors use second harmonic generation analysis and molecular dynamics simulations to reveal that clinical IDO1 inhibitors differentially modulate its signaling, impacting distinct transcriptomes.

The tryptophan-degrading enzyme indoleamine 2,3-dioxygenase 1 (IDO1) is a plastic immune checkpoint molecule that potently orchestrates immune responses within the tumor microenvironment (TME). As a heme-containing protein, IDO1 catalyzes the conversion of the essential amino acid tryptophan into immunoactive metabolites, called kynurenines. By depleting tryptophan and enriching the TME with kynurenines, IDO1 catalytic activity shapes an immunosuppressive TME. Accordingly, the inducible or constitutive IDO1 expression in cancer correlates with a negative prognosis for patients, representing one of the critical tumor-escape mechanisms. However, clinically trialed IDO1 catalytic inhibitors disappointed the expected anti-tumor efficacy. Interestingly, the non-enzymatic apo-form of IDO1 is still active as a transducing protein, capable of promoting an immunoregulatory phenotype in dendritic cells (DCs) as well as a pro-tumorigenic behavior in murine melanoma. Moreover, the IDO1 catalytic inhibitor epacadostat can induce a tolerogenic phenotype in plasmacytoid DCs, overcoming the catalytic inhibition of IDO1. Based on this recent evidence, IDO1 plasticity was investigated in the human ovarian cancer cell line, SKOV-3, that constitutively expresses IDO1 in a dynamic balance between the holo- and apo-protein, and thus potentially endowed with a dual function (i.e., enzymatic and non-enzymatic). Besides inhibiting the catalytic activity, epacadostat persistently stabilizes the apo-form of IDO1 protein, favoring its tyrosine-phosphorylation and promoting its association with the phosphatase SHP-2. In SKOV-3 cells, both these early molecular events activate a signaling pathway transduced by IDO1 apo-protein, which is independent of its catalytic activity and contributes to the tumorigenic phenotype of SKOV-3 cells. Overall, our findings unveiled a new mechanism of action of epacadostat on IDO1 target, repositioning the catalytic inhibitor as a stabilizer of the apo-form of IDO1, still capable of transducing a pro-tumorigenic pathway in SKOV-3 tumor. This mechanism could contribute to clarify the lack of effectiveness of epacadostat in clinical trials and shed light on innovative immunotherapeutic strategies to tackle IDO1 target.

Cellulosic fabrics made of cotton, linen, bamboo, hemp, and nettle, were selected for the eco-sustainable production of colored biofunctional textiles, using natural dyes from onion skin of the Dorata di Parma (Allium cepa L.) variety. Dyeing experiments were performed in net water with and without pre-treatment with alum, tin chloride, and tannic acid as mordants. The color and the UV-protection factor (UPF) were evaluated through UV-visible spectroscopy. The treatment with onion skin extract induced a relevant increase of UPF in all investigated fabrics, promoting the protection category from insufficient (UPF<15) to good for linen (UPF = 21-26), up to very good and excellent for cotton (UPF = 35-66) and bamboo (UPF = 48-56), respectively. To mimic the direct contact with the skin, textile samples were immersed in artificial sweat where dyed cotton, hemp, and nettle were found to promote higher phenolic release (0.28-0.50 mg gallic acid equivalents/g textile) than linen and bamboo (0.12-0.22 mg gallic acid equivalents/g textile). The obtained "biofunctional sweat" did not impact on the survival of primary human dermal fibroblasts (HDF) and skin keratinocytes (NCTC2455), while protected these cells against heat shock and reduced reactive oxygen species levels upon exposure to pro-oxidant and inflammatory agents as H2O2 and lipopolysaccharide, respectively. Increased activity levels of the H2O2-scavenging enzyme Catalase (CAT) were also observed, thus demonstrating enhanced stress response and homeostatic capability of these cells. This study provides evidence that extracts from onion waste can be used to functionalize cellulosic fabrics conferring properties as natural shield against UV radiation, and antioxidant and anti-inflammatory materials thus holding potential in skin protection and prevention of dermatological diseases.

Influenza viruses (IV) are single-stranded RNA viruses with a negative-sense genome and have the potential to cause pandemics. While vaccines exist for influenza, their protection is only partial. Additionally, there is only a limited number of approved anti-IV drugs, which are associated to emergence of drug resistance. To address these issues, for years we have focused on the development of small-molecules that can interfere with the heterodimerization of PA and PB1 subunits of the IV RNA-dependent RNA polymerase (RdRP). In this study, starting from a cycloheptathiophene-3-carboxamide compound that we recently identified, we performed iterative cycles of medicinal chemistry optimization that led to the identification of compounds 43 and 45 with activity in the nanomolar range against circulating A and B strains of IV. Mechanistic studies demonstrated the ability of 43 and 45 to interfere with viral RdRP activity by disrupting PA-PB1 subunits heterodimerization and to bind to the PA C-terminal domain through biophysical assays. Most important, ADME studies of 45 also showed an improvement in the pharmacokinetic profile with respect to the starting hit.

Indoleamine 2,3-dioxygenase 1 (IDO1) plays a key role in tumor immune escape. Besides being a metabolic enzyme that catalyzes the first step of tryptophan catabolism, it also acts as a signal-transducing protein, whose partnering with tyrosine phosphatase Src homology 2 (SH2) domain-containing protein tyrosine phosphatase substrate (SHPs) and phosphatidylinositol-3-kinase (PI3K) regulatory subunit p85 promotes the establishment of a sustained immunosuppressive phenotype. While IDO1 inhibitors typically interfere with its enzymatic activity, we aimed to discover a more effective modulator capable of blocking not only the enzymatic but also the signaling-mediated functions of IDO1. By virtual screening, we identified the compound VS-15, which selectively binds the heme-free form of IDO1, inhibits its enzymatic activity, and reduces the IDO1-mediated signaling pathway by negatively interfering with its partnership with SHPs and PI3K regulatory subunit p85 as well as with the IDO1 anchoring to the early endosomes in tumor cells. Moreover, VS-15 counteracts the TGF-β-mediated immunosuppressive phenotype in dendritic cells and reduces the level of inhibition of T cell proliferation by suppressive monocytes isolated from patients affected by pancreatic cancer. Herein, we describe the discovery and characterization of a small molecule with an unprecedented mechanism of action, capable of inhibiting both the enzymatic and nonenzymatic activities of IDO1 by binding to its apo-form. These results pave the way for the development of next-generation IDO1 inhibitors with a unique competitive advantage over the currently available modulators, thereby opening therapeutic opportunities in cancer immunotherapy.

Allantoin represents a compound widely employed in pharmaceutical and cosmetic fields. Its safety has been acknowledged by regulatory bodies such as the US Food and Drug Administration, the European Commission for Cosmetics and Consumer and Health and European Directorate for the Quality of Medicines & HealthCare. This justifies its wide use in dermatological/cosmetic formulations and allows their safe use.Allantoin possesses an asymmetric carbon atom, resulting in two enantiomers, with the (S)-enantiomer predominating in plants, although racemization may potentially occur during manufacturing processes. Notably, literature currently lacks enantioselective LC methods for allantoin analysis.In this study, two zwitterionic Cinchona alkaloid-based chiral stationary phases (CSPs), commercially known as CHIRALPAK® ZWIX(+) (CSP1) and CHIRALPAK® ZWIX(-) (CSP2), were utilized for the enantioseparation of allantoin under polar-ionic conditions. By employing a mobile phase consisting of acetonitrile/methanol/water/acetic acid (96:2:2:0.1, v/v/v/v), nearly complete baseline separation (with α=1.08) of allantoin enantiomers was achieved in less than 15 min with both CSPs. Due to the “pseudo-enantiomeric” nature of the two chiral selectors (quinine-based in CSP1 and quinidine-based in CSP2), an inversion of the enantiomer elution order was observed with the two CSPs under identical experimental conditions. Remarkably, this represents a rare instance where these CSPs demonstrate the ability to enantioseparate a non-ionic, non-ionizable species.The application of a molecular dynamics in silico protocol proved useful in elucidating the retention mechanism in depth, casting light on the central role of the H-bond formation and the involvement of the anionic moiety of the CSP 1.

Multiple sclerosis is a debilitating autoimmune disease, characterized by chronic inflammation of the central nervous system. While the significance of the gut microbiome on multiple sclerosis pathogenesis is established, the underlining mechanisms are unknown. We found that serum levels of the microbial postbiotic tryptophan metabolite indole-3-carboxaldehyde (3-IAld) inversely correlated with disease duration in multiple sclerosis patients. Much like the host-derived tryptophan derivative L-Kynurenine, 3-IAld would bind and activate the Aryl hydrocarbon Receptor (AhR), which, in turn, controls endogenous tryptophan catabolic pathways. As a result, in peripheral lymph nodes, microbial 3-IAld, affected mast-cell tryptophan metabolism, forcing mast cells to produce serotonin via Tph1. We thus propose a protective role for AhR-mast-cell activation driven by the microbiome, whereby natural metabolites or postbiotics will have a physiological role in immune homeostasis and may act as therapeutic targets in autoimmune diseases.

In recent years, the restoration of p53 physiological functions has become an attractive therapeutic approach to develop novel and efficacious cancer therapies. Among other mechanisms, the oncosuppressor protein p53 is functionally regulated by MDM2 through its E3 ligase function. MDM2 promotes p53 ubiquitination and degradation following homodimerization or heterodimerization with MDM4. Recently, we discovered Pep3 (1, Pellegrino et al., 2015), a novel peptidic inhibitor of MDM2 dimerization able to restore p53 oncosuppressive functions both in vitro and in vivo. In this work, we were able to identify the key interactions between peptide 1 and MDM2 RING domain and to design peptide 2, a truncated version of 1 that is still able to bind MDM2. Integrating both computational and biophysical techniques, we show that peptide 2 maintains the conserved peptide 1-MDM2 interactions and is still able to bind to full-length MDM2.

Dimethylarginine dimethylaminohydrolase-1 (DDAH-1) accounts for the catabolism of the endogenous inhibitors of nitric oxide (NO) synthases, namely, ADMA (Nω,Nω-dimethyl-l-arginine) and NMMA (Nω-monomethyl-l-arginine). Inhibition of DDAH-1 may prove a therapeutic benefit in diseases associated with elevated nitric oxide (NO) levels by providing a tissue-specific increase of ADMA and NMMA. In this work, we have used molecular dynamics to generate a pool of DDAH-1 conformations in the apo and holo forms. Ensemble docking has been instrumental in screening an in-house fragment-based library of 824 compounds. Resulting virtual hits have been validated for their binding activity to recombinant human DDAH-1 using microscale thermophoresis (MST). As a key result, three non-amino acidic ligands of DDAH-1 (VIS212, VIS268, VIS726) are identified with higher binding efficiency index than ADMA. Amid these compounds, purpurogallin (VIS726) proves a potent ligand of DDAH-1, showing a mixed behavior of enzymatic inhibition in a biochemical assay. This finding widens the panel of known molecular targets of purpurogallin and provides clues into the molecular mechanisms of its cellular NO inhibition activity as well as its anti-inflammatory and neuroprotective effects.