Fungal and bacterial species richness in biodeteriorated seventeenth century Venetian manuscripts.

Historical paper documents are susceptible to complex degradation processes, including biodeterioration, which can progressively compromise their aesthetic and structural integrity. This study analyses seventeenth century handwritten historical letters stored at the Correr Museum Library in Venice, Italy, exhibiting pronounced signs of biodeterioration. The techniques used encompassed traditional colony isolation on agar plates and proteomics analyses, employing nanoscale liquid chromatography coupled with high-resolution mass spectrometry (nano-LC-MS). Fluorescence microscopy was used for the first time in the historical paper biodeterioration context to supplement the conventional stereoscopic, optical, and scanning electron microscopic imaging techniques. This method enables the visualisation of microorganisms beyond and beneath the paper's surface through their natural intrinsic autofluorescence in a non-invasive and non-destructive way. The results demonstrate a diverse, complex, and abundant microbiota composed of coexisting fungal and bacterial species (Ascomycota, Mucoromycota, Basidiomycota, Proteobacteria, and Actinobacteria), along with mite carcasses, insects, parasites, and possibly protists. Furthermore, this study reveals certain species that were not previously documented in the biodeterioration of historical paper, including human pathogens, such as Histoplasma capsulatum, Brucella, Candida albicans, and species of Aspergillus (A. flavus, A. fumigatus, A. oryzae, A. terreus, A. niger) known to cause infections or produce mycotoxins, posing substantial risk to both artefacts and humans.

Computer-aided identification, synthesis, and biological evaluation of DNA polymerase η inhibitors for the treatment of cancer.

In cancer cells, Pol η allows DNA replication and cell proliferation even in the presence of chemotherapeutic drug-induced damages, like in the case of platinum-containing drugs. Inhibition of Pol η thus represents a promising strategy to overcome drug resistance and preserve the effectiveness of chemotherapeutic drugs. Here, we report the discovery of a novel class of Pol ƞ inhibitors, with 35 active close analogs. Compound 21 (ARN24964) stands out as the best inhibitor, with an IC50 value of 14.7 µM against Pol η and a good antiproliferative activity when used in combination with cisplatin - with a synergistic effect in three different cancer cell lines (A375, A549, OVCAR3). Moreover, it is characterized by a favorable drug-like profile in terms of its aqueous kinetic solubility, plasma and metabolic stability. Thus, ARN24964 is a promising compound for further structure-based drug design efforts toward developing drugs to solve or limit the issue of drug resistance to platinum-containing drugs in cancer patients.

Design, synthesis, docking, and biochemical characterization of non-nucleoside SARS-CoV-2 RdRp inhibitors.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide pandemic. The identification of effective antiviral drugs remains an urgent medical need. In this context, here we report 17 new 1,4-benzopyrone derivatives, which have been designed, synthesized, and characterized for their ability to block the RNA-dependent RNA polymerase (RdRp) enzyme, a promising target for antiviral drug discovery. This compound series represents a good starting point for developing non-nucleoside inhibitors of RdRp. Compounds 4, 5, and 8 were the most promising drug-like candidates with good potency in inhibiting RdRp, improved in vitro pharmacokinetics compared to the initial hits, and no cytotoxicity effects on normal cell (HEK-293). Compound 8 (ARN25592) stands out as the most promising inhibitor. Our results indicate that this new chemical class of 1,4-benzopyrone derivatives deserves further exploration towards novel and potent antiviral drugs for the treatment of SARS-CoV-2 and potentially other viruses.

Quercetin and luteolin are single-digit micromolar inhibitors of the SARS-CoV-2 RNA-dependent RNA polymerase.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly become a global health pandemic. Among the viral proteins, RNA-dependent RNA polymerase (RdRp) is responsible for viral genome replication and has emerged as one of the most promising targets for pharmacological intervention against SARS-CoV-2. To this end, we experimentally tested luteolin and quercetin for their ability to inhibit the RdRp enzyme. These two compounds are ancestors of flavonoid natural compounds known for a variety of basal pharmacological activities. Luteolin and quercetin returned a single-digit IC50 of 4.6 µM and 6.9 µM, respectively. Then, through dynamic docking simulations, we identified possible binding modes of these compounds to a recently published cryo-EM structure of RdRp. Collectively, these data indicate that these two compounds are a valid starting point for further optimization and development of a new class of RdRp inhibitors to treat SARS-CoV-2 and potentially other viral infections.

Design, Synthesis, In Vitro and In Vivo Characterization of Selective NKCC1 Inhibitors for the Treatment of Core Symptoms in Down Syndrome.

Intracellular chloride concentration [Cl-]i is defective in several neurological disorders. In neurons, [Cl-]i is mainly regulated by the action of the Na+-K+-Cl- importer NKCC1 and the K+-Cl- exporter KCC2. Recently, we have reported the discovery of ARN23746 as the lead candidate of a novel class of selective inhibitors of NKCC1. Importantly, ARN23746 is able to rescue core symptoms of Down syndrome (DS) and autism in mouse models. Here, we describe the discovery and extensive characterization of this chemical class of selective NKCC1 inhibitors, with focus on ARN23746 and other promising derivatives. In particular, we present compound 40 (ARN24092) as a backup/follow-up lead with in vivo efficacy in a mouse model of DS. These results further strengthen the potential of this new class of compounds for the treatment of core symptoms of brain disorders characterized by the defective NKCC1/KCC2 expression ratio.

Design, Synthesis, and Biological Evaluation of a Series of Oxazolone Carboxamides as a Novel Class of Acid Ceramidase Inhibitors.

Acid ceramidase (AC) is a cysteine hydrolase that plays a crucial role in the metabolism of lysosomal ceramides, important members of the sphingolipid family, a diversified class of bioactive molecules that mediate many biological processes ranging from cell structural integrity, signaling, and cell proliferation to cell death. In the effort to expand the structural diversity of the existing collection of AC inhibitors, a novel class of substituted oxazol-2-one-3-carboxamides were designed and synthesized. Herein, we present the chemical optimization of our initial hits, 2-oxo-4-phenyl-N-(4-phenylbutyl)oxazole-3-carboxamide 8a and 2-oxo-5-phenyl-N-(4-phenylbutyl)oxazole-3-carboxamide 12a, which resulted in the identification of 5-[4-fluoro-2-(1-methyl-4-piperidyl)phenyl]-2-oxo-N-pentyl-oxazole-3-carboxamide 32b as a potent AC inhibitor with optimal physicochemical and metabolic properties, showing target engagement in human neuroblastoma SH-SY5Y cells and a desirable pharmacokinetic profile in mice, following intravenous and oral administration. 32b enriches the arsenal of promising lead compounds that may therefore act as useful pharmacological tools for investigating the potential therapeutic effects of AC inhibition in relevant sphingolipid-mediated disorders.

Identification, Structure-Activity Relationship, and Biological Characterization of 2,3,4,5-Tetrahydro-1H-pyrido[4,3-b]indoles as a Novel Class of CFTR Potentiators.

Cystic fibrosis (CF) is a life-threatening autosomal recessive disease, caused by mutations in the CF transmembrane conductance regulator (CFTR) chloride channel. CFTR modulators have been reported to address the basic defects associated with CF-causing mutations, partially restoring the CFTR function in terms of protein processing and/or channel gating. Small-molecule compounds, called potentiators, are known to ameliorate the gating defect. In this study, we describe the identification of the 2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole core as a novel chemotype of potentiators. In-depth structure-activity relationship studies led to the discovery of enantiomerically pure 39 endowed with a good efficacy in rescuing the gating defect of F508del- and G551D-CFTR and a promising in vitro druglike profile. The in vivo characterization of γ-carboline 39 showed considerable exposure levels and good oral bioavailability, with detectable distribution to the lungs after oral administration to rats. Overall, these findings may represent an encouraging starting point to further expand this chemical class, adding a new chemotype to the existing classes of CFTR potentiators.

Multitarget Compounds for Bipolar Disorder: From Rational Design to Preliminary Pharmacokinetic Evaluation.

Due to the complex and multifactorial nature of bipolar disorder (BD), single-target drugs have traditionally provided limited relief with no disease-modifying effects. In line with the polypharmacology paradigm, we attempted to overcome these limitations by devising two series of multitarget-directed ligands endowed with both a partial agonist profile at dopamine receptor D3 (D3R) and inhibitory activity against glycogen synthase kinase 3 beta (GSK-3ß). These are two structurally unrelated targets that play independent, yet connected, roles in cognition and mood regulation. Two compounds (7 and 10) emerged as promising D3R/GSK-3ß multitarget-directed ligands with nanomolar activity at D3R and low-micromolar inhibition of GSK-3ß, thereby confirming, albeit preliminarily, the feasibility of our strategy. Furthermore, 7 showed promising drug-like properties in stability and pharmacokinetic studies.

Design, Synthesis, Dynamic Docking, Biochemical Characterization, and in Vivo Pharmacokinetics Studies of Novel Topoisomerase II Poisons with Promising Antiproliferative Activity.

We previously reported a first set of hybrid topoisomerase II (topoII) poisons whose chemical core merges key pharmacophoric elements of etoposide and merbarone, which are two well-known topoII blockers. Here, we report on the expansion of this hybrid molecular scaffold and present 16 more hybrid derivatives that have been designed, synthesized, and characterized for their ability to block topoII and for their overall drug-like profile. Some of these compounds act as topoII poison and exhibit good solubility, metabolic (microsomal) stability, and promising cytotoxicity in three cancer cell lines (DU145, HeLa, A549). Compound 3f (ARN24139) is the most promising drug-like candidate, with a good pharmacokinetics profile in vivo. Our results indicate that this hybrid new chemical class of topoII poisons deserves further exploration and that 3f is a favorable lead candidate as a topoII poison, meriting future studies to test its efficacy in in vivo tumor models.

Lead Optimization of Benzoxazolone Carboxamides as Orally Bioavailable and CNS Penetrant Acid Ceramidase Inhibitors.

Sphingolipids (SphLs) are a diverse class of molecules that are regulated by a complex network of enzymatic pathways. A disturbance in these pathways leads to lipid accumulation and initiation of several SphL-related disorders. Acid ceramidase is one of the key enzymes that regulate the metabolism of ceramides and glycosphingolipids, which are important members of the SphL family. Herein, we describe the lead optimization studies of benzoxazolone carboxamides resulting in piperidine 22m, where we demonstrated target engagement in two animal models of neuropathic lysosomal storage diseases (LSDs), Gaucher's and Krabbe's diseases. After daily intraperitoneal administration at 90 mg kg-1, 22m significantly reduced the brain levels of the toxic lipids glucosylsphingosine (GluSph) in 4L;C* mice and galactosylsphingosine (GalSph) in Twitcher mice. We believe that 22m is a lead molecule that can be further developed for the correction of severe neurological LSDs where GluSph or GalSph play a significant role in disease pathogenesis.

Inhibition of Serine Palmitoyltransferase by a Small Organic Molecule Promotes Neuronal Survival after Astrocyte Amyloid Beta 1-42 Injury.

Alzheimer's disease (AD) is a slow-progressing disease of the brain characterized by symptoms such as impairment of memory and other cognitive functions. AD is associated with an inflammatory process that involves astrocytes and microglial cells, among other components. Astrocytes are the most abundant type of glial cells in the central nervous system (CNS). They are involved in inducing neuroinflammation. The present study uses astrocyte-neuron cocultures to investigate how ARN14494, a serine palmitoyltransferase (SPT) inhibitor, affects the CNS in terms of anti-inflammation and neuroprotection. SPT is the first rate-limiting enzyme in the de novo ceramide synthesis pathway. Consistent evidence suggests that ceramide is increased in AD brain patients. After ß-amyloid 1-42 injury in an in vitro model of AD, ARN14494 inhibits SPT activity and the synthesis of long-chain ceramides and dihydroceramides that are involved in AD progression. In mouse primary cortical astrocytes, ARN14494 prevents the synthesis of proinflammatory cytokines TNFα and IL1ß, growth factor TGFß1, and oxidative stress-related enzymes iNOS and COX2. ARN14494 also exerts neuroprotective properties in primary cortical neurons. ARN14494 decreases neuronal death and caspase-3 activation in neurons, when the neuroinflammation is attenuated in astrocytes. These findings suggest that ARN14494 protects neurons from ß-amyloid 1-42 induced neurotoxicity through a variety of mechanisms, including antioxidation, antiapoptosis, and anti-inflammation. SPT inhibition could therefore be a safe therapeutic strategy for ameliorating the pathology of Alzheimer's disease.

A New Drug Delivery System Based on Tauroursodeoxycholic Acid and PEDOT.

Localized drug delivery represents one of the most challenging uses of systems based on conductive polymer films. Typically, anionic drugs are incorporated within conductive polymers through electrostatic interaction with the positively charged polymer. Following this approach, the synthetic glucocorticoid dexamethasone phosphate is often delivered from neural probes to reduce the inflammation of the surrounding tissue. In light of the recent literature on the neuroprotective and anti-inflammatory properties of tauroursodeoxycholic acid (TUDCA), for the first time, this natural bile acid was incorporated within poly(3,4-ethylenedioxythiophene) (PEDOT). The new material, PEDOT-TUDCA, efficiently promoted an electrochemically controlled delivery of the drug, while preserving optimal electrochemical properties. Moreover, the low cytotoxicity observed with viability assays, makes PEDOT-TUDCA a good candidate for prolonging the time span of chronic neural recording brain implants.

Biochemically Controlled Release of Dexamethasone Covalently Bound to PEDOT.

PEDOT (Poly(3,4-ethylenedioxythiophene)) is one of the most promising electrode materials for biomedical applications like neural recording and stimulation, thanks to its enhanced biocompatibility and electronic properties. Drug delivery by PEDOT is typically achieved by incorporating drugs as dopants during the electrodeposition procedure and a subsequent release can be promoted by applying a cathodic trigger that reduces PEDOT while enabling the drug to diffuse. This approach has several disadvantages including, for instance, the release of contaminants mainly due to PEDOT decomposition during electrochemical release. Herein we describe a new strategy based on the formation of a chemical linkage between the drug and the conductive polymer. In particular, dexamethasone was successfully integrated into a new electropolymerized PEDOT-Dex composite, leading to a self-adjusting drug release system based on a biochemically hydrolysable bond between dexamethasone and PEDOT.

Design, Synthesis, Structure-Activity Relationship Studies, and Three-Dimensional Quantitative Structure-Activity Relationship (3D-QSAR) Modeling of a Series of O-Biphenyl Carbamates as Dual Modulators of Dopamine D3 Receptor and Fatty Acid Amide Hydrolase.

We recently reported molecules designed according to the multitarget-directed ligand paradigm to exert combined activity at human fatty acid amide hydrolase (FAAH) and dopamine receptor subtype D3 (D3R). Both targets are relevant for tackling several types of addiction (most notably nicotine addiction) and other compulsive behaviors. Here, we report an SAR exploration of a series of biphenyl-N-[4-[4-(2,3-substituted-phenyl)piperazine-1-yl]alkyl]carbamates, a novel class of molecules that had shown promising activities at the FAAH-D3R target combination in preliminary studies. We have rationalized the structural features conducive to activities at the main targets and investigated activities at two off-targets: dopamine receptor subtype D2 and endocannabinoid receptor CB1. To understand the unexpected affinity for the CB1 receptor, we devised a 3D-QSAR model, which we then prospectively validated. Compound 33 was selected for PK studies because it displayed balanced affinities for the main targets and clear selectivity over the two off-targets. 33 has good stability and oral bioavailability and can cross the blood-brain barrier.

Kernel-Based, Partial Least Squares Quantitative Structure-Retention Relationship Model for UPLC Retention Time Prediction: A Useful Tool for Metabolite Identification.

We propose a new QSRR model based on a Kernel-based partial least-squares method for predicting UPLC retention times in reversed phase mode. The model was built using a combination of classical (physicochemical and topological) and nonclassical (fingerprints) molecular descriptors of 1383 compounds, encompassing different chemical classes and structures and their accurately measured retention time values. Following a random splitting of the data set into a training and a test set, we tested the ability of the model to predict the retention time of all the compounds. The best predicted/experimental R2 value was higher than 0.86, while the best Q2 value we observed was close to 0.84. A comparison of our model with traditional and simpler MLR and PLS regression models shows that KPLS better performs in term of correlation (R2), prediction (Q2), and support to MetID peak assignment. The KPLS model succeeded in two real-life MetID tasks by correctly predicting elution order of Phase I metabolites, including isomeric monohydroxylated compounds. We also show in this paper that the model's predictive power can be extended to different gradient profiles, by simple mathematical extrapolation using a known equation, thus offering very broad flexibility. Moreover, the current study includes a deep investigation of different types of chemical descriptors used to build the structure-retention relationship.

Second-Generation Non-Covalent NAAA Inhibitors are Protective in a Model of Multiple Sclerosis.

Palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) are endogenous lipid mediators that suppress inflammation. Their actions are terminated by the intracellular cysteine amidase, N-acylethanolamine acid amidase (NAAA). Even though NAAA may offer a new target for anti-inflammatory therapy, the lipid-like structures and reactive warheads of current NAAA inhibitors limit the use of these agents as oral drugs. A series of novel benzothiazole-piperazine derivatives that inhibit NAAA in a potent and selective manner by a non-covalent mechanism are described. A prototype member of this class (8) displays high oral bioavailability, access to the central nervous system (CNS), and strong activity in a mouse model of multiple sclerosis (MS). This compound exemplifies a second generation of non-covalent NAAA inhibitors that may be useful in the treatment of MS and other chronic CNS disorders.

Potent α-amino-ß-lactam carbamic acid ester as NAAA inhibitors. Synthesis and structure-activity relationship (SAR) studies.

4-Cyclohexylbutyl-N-[(S)-2-oxoazetidin-3-yl]carbamate (3b) is a potent, selective and systemically active inhibitor of intracellular NAAA activity, which produces profound anti-inflammatory effects in animal models. In the present work, we describe structure-activity relationship (SAR) studies on 3-aminoazetidin-2-one derivatives, which have led to the identification of 3b, and expand these studies to elucidate the principal structural and stereochemical features needed to achieve effective NAAA inhibition. Investigations on the influence of the substitution at the ß-position of the 2-oxo-3-azetidinyl ring as well as on the effect of size and shape of the carbamic acid ester side chain led to the discovery of 3ak, a novel inhibitor of human NAAA that shows an improved physicochemical and drug-like profile relative to 3b. This favourable profile, along with the structural diversity of the carbamic acid chain of 3b, identify this compound as a promising new tool to investigate the potential of NAAA inhibitors as therapeutic agents for the treatment of pain and inflammation.

Fluorine nuclear magnetic resonance-based assay in living mammalian cells.

Nuclear magnetic resonance (NMR)-based screening has been recognized as a powerful approach for the identification and characterization of molecules interacting with pharmaceutical targets. Indeed, several NMR methods have been developed and successfully applied to many drug discovery projects. Whereas most of these approaches have targeted isolated biomolecular receptors, very few cases are reported with the screening performed in intact cells and cell extracts. Here we report the first successful application of the fluorine NMR-based assay n-FABS (n-fluorine atoms for biochemical screening) in living mammalian cells expressing the membrane protein fatty acid amide hydrolase (FAAH). This method allows the identification of both weak and potent inhibitors and the measurement of their potency in a physiological environment.

Hit Optimization of 5-Substituted-N-(piperidin-4-ylmethyl)-1H-indazole-3-carboxamides: Potent Glycogen Synthase Kinase-3 (GSK-3) Inhibitors with in Vivo Activity in Model of Mood Disorders.

Novel treatments for bipolar disorder with improved efficacy and broader spectrum of activity are urgently needed. Glycogen synthase kinase 3ß (GSK-3ß) has been suggested to be a key player in the pathophysiology of bipolar disorder. A series of novel GSK-3ß inhibitors having the common N-[(1-alkylpiperidin-4-yl)methyl]-1H-indazole-3-carboxamide scaffold were prepared taking advantage of an X-ray cocrystal structure of compound 5 with GSK-3ß. We probed different substitutions at the indazole 5-position and at the piperidine-nitrogen to obtain potent ATP-competitive GSK-3ß inhibitors with good cell activity. Among the compounds assessed in the in vivo PK experiments, 14i showed, after i.p. dosing, encouraging plasma PK profile and brain exposure, as well as efficacy in a mouse model of mania. Compound 14i was selected for further in vitro/in vivo pharmacological evaluation, in order to elucidate the use of ATP-competitive GSK-3ß inhibitors as new tools in the development of new treatments for mood disorders.

O-(triazolyl)methyl carbamates as a novel and potent class of fatty acid amide hydrolase (FAAH) inhibitors.

Inhibition of fatty acid amide hydrolase (FAAH) activity is under investigation as a valuable strategy for the treatment of several disorders, including pain and drug addiction. A number of potent FAAH inhibitors belonging to different chemical classes have been disclosed to date; O-aryl carbamates are one of the most representative families. In the search for novel FAAH inhibitors, a series of O-(1,2,3-triazol-4-yl)methyl carbamate derivatives were designed and synthesized exploiting a copper- catalyzed [3+2] cycloaddition reaction between azides and alkynes (click chemistry). Exploration of the structure-activity relationships within this new class of compounds identified potent inhibitors of both rat and human FAAH with IC50 values in the single-digit nanomolar range. In addition, these derivatives showed improved stability in rat plasma and kinetic solubility in buffer with respect to the lead compound. Based on the results of the study, the novel analogues identified can be considered to be promising starting point for the development of new FAAH inhibitors with improved drug-like properties.