Extraction and Quantification of Azelaic Acid from Different Wheat Samples (Triticum durum Desf.) and Evaluation of Their Antimicrobial and Antioxidant Activities.

Azelaic Acid (AzA) is a 9-carbon atom dicarboxylic acid, with numerous pharmacological uses in dermatology. Its effectiveness in papulopustular rosacea and acne vulgaris, among other dermatological disorders such as keratinization and hyper-pigmentation, is thought to be related to its anti-inflammatory and antimicrobial properties. It is a by-product of Pityrosporum fungal mycelia metabolism but also it is found in different cereals such as barley, wheat, and rye. Diverse topical formulations of AzA exist in commerce, and it is mainly produced via chemical synthesis. In this study we describe the extraction of AzA from whole grains and whole-grain flour (Triticum durum Desf.) through green methods. Seventeen different extracts were prepared and analyzed for their AzA content by HPLC-MS methods and then screened for their antioxidant activity using spectrophotometric assays (ABTS, DPPH, and Folin-Ciocalteu). Minimum-inhibitory-concentration (MIC) assays against several bacterial and fungal pathogens were performed, to validate their antimicrobial activity. The obtained results indicate that whole grain extracts provide a wider spectrum of activity than the flour matrix; in particular, the Naviglio® extract showed higher AzA content, while the hydroalcoholic ultrasound-assisted extract provided better antimicrobial and antioxidant activity. The data analysis was performed using principal component analysis (PCA), as an unsupervised-pattern-recognition technique, to extract useful analytical and biological information.

Cycloserine enantiomers are reversible inhibitors of human alanine:glyoxylate aminotransferase: implications for Primary Hyperoxaluria type 1.

Peroxisomal alanine:glyoxylate aminotransferase (AGT) is responsible for glyoxylate detoxification in human liver and utilizes pyridoxal 5'-phosphate (PLP) as coenzyme. The deficit of AGT leads to Primary Hyperoxaluria Type I (PH1), a rare disease characterized by calcium oxalate stones deposition in the urinary tract as a consequence of glyoxylate accumulation. Most missense mutations cause AGT misfolding, as in the case of the G41R, which induces aggregation and proteolytic degradation. We have investigated the interaction of wild-type AGT and the pathogenic G41R variant with d-cycloserine (DCS, commercialized as Seromycin), a natural product used as a second-line treatment of multidrug-resistant tuberculosis, and its synthetic enantiomer l-cycloserine (LCS). In contrast with evidences previously reported on other PLP-enzymes, both ligands are AGT reversible inhibitors showing inhibition constants in the micromolar range. While LCS undergoes half-transamination generating a ketimine intermediate and behaves as a classical competitive inhibitor, DCS displays a time-dependent binding mainly generating an oxime intermediate. Using a mammalian cellular model, we found that DCS, but not LCS, is able to promote the correct folding of the G41R variant, as revealed by its increased specific activity and expression as a soluble protein. This effect also translates into an increased glyoxylate detoxification ability of cells expressing the variant upon treatment with DCS. Overall, our findings establish that DCS could play a role as pharmacological chaperone, thus suggesting a new line of intervention against PH1 based on a drug repositioning approach. To a widest extent, this strategy could be applied to other disease-causing mutations leading to AGT misfolding.

Refining the structure-activity relationships of 2-phenylcyclopropane carboxylic acids as inhibitors of O-acetylserine sulfhydrylase isoforms.

The lack of efficacy of current antibacterials to treat multidrug resistant bacteria poses a life-threatening alarm. In order to develop enhancers of the antibacterial activity, we carried out a medicinal chemistry campaign aiming to develop inhibitors of enzymes that synthesise cysteine and belong to the reductive sulphur assimilation pathway, absent in mammals. Previous studies have provided a novel series of inhibitors for O-acetylsulfhydrylase - a key enzyme involved in cysteine biosynthesis. Despite displaying nanomolar affinity, the most active representative of the series was not able to interfere with bacterial growth, likely due to poor permeability. Therefore, we rationally modified the structure of the hit compound with the aim of promoting their passage through the outer cell membrane porins. The new series was evaluated on the recombinant enzyme from Salmonella enterica serovar Typhimurium, with several compounds able to keep nanomolar binding affinity despite the extent of chemical manipulation.

Strategies to Overcome Antimicrobial Resistance (AMR) Making Use of Non-Essential Target Inhibitors: A Review.

Antibiotics have always been considered as one of the most relevant discoveries of the twentieth century. Unfortunately, the dawn of the antibiotic era has sadly corresponded to the rise of the phenomenon of antimicrobial resistance (AMR), which is a natural process whereby microbes evolve in such a way to withstand the action of drugs. In this context, the identification of new potential antimicrobial targets and/or the identification of new chemical entities as antimicrobial drugs are in great demand. To date, among the many possible approaches used to deal with antibiotic resistance is the use of antibiotic adjuvants that hit bacterial non-essential targets. In this review, the author focuses on the discovery of antibiotic adjuvants and on new tools to study and reduce the prevalence of resistant bacterial infections.

Integration of Enhanced Sampling Methods with Saturation Transfer Difference Experiments to Identify Protein Druggable Pockets.

Saturation transfer difference (STD) is an NMR technique conventionally applied in drug discovery to identify ligand moieties relevant for binding to protein cavities. This is important to direct medicinal chemistry efforts in small-molecule optimization processes. However, STD does not provide any structural details about the ligand-target complex under investigation. Herein, we report the application of a new integrated approach, which combines enhanced sampling methods with STD experiments, for the characterization of ligand-target complexes that are instrumental for drug design purposes. As an example, we have studied the interaction between StOASS-A, a potential antibacterial target, and an inhibitor previously reported. This approach allowed us to consider the ligand-target complex from a dynamic point of view, revealing the presence of an accessory subpocket which can be exploited to design novel StOASS-A inhibitors. As a proof of concept, a small library of derivatives was designed and evaluated in vitro, displaying the expected activity.

Discovering a new class of antifungal agents that selectively inhibits microbial carbonic anhydrases.

Infections caused by pathogens resistant to the available antimicrobial treatments represent nowadays a threat to global public health. Recently, it has been demonstrated that carbonic anhydrases (CAs) are essential for the growth of many pathogens and their inhibition leads to growth defects. Principal drawbacks in using CA inhibitors (CAIs) as antimicrobial agents are the side effects due to the lack of selectivity toward human CA isoforms. Herein we report a new class of CAIs, which preferentially interacts with microbial CA active sites over the human ones. The mechanism of action of these inhibitors was investigated against an important fungal pathogen, Cryptococcus neoformans, revealing that they are also able to inhibit CA in microbial cells growing in vitro. At our best knowledge, this is the first report on newly designed synthetic compounds selectively targeting ß-CAs and provides a proof of concept of microbial CAs suitability as an antimicrobial drug target.

Discovery of novel fragments inhibiting O-acetylserine sulphhydrylase by combining scaffold hopping and ligand-based drug design.

Several bacteria rely on the reductive sulphur assimilation pathway, absent in mammals, to synthesise cysteine. Reduction of virulence and decrease in antibiotic resistance have already been associated with mutations on the genes that codify cysteine biosynthetic enzymes. Therefore, inhibition of cysteine biosynthesis has emerged as a promising strategy to find new potential agents for the treatment of bacterial infection. Following our previous efforts to explore OASS inhibition and to expand and diversify our library, a scaffold hopping approach was carried out, with the aim of identifying a novel fragment for further development. This novel chemical tool, endowed with favourable pharmacological characteristics, was successfully developed, and a preliminary Structure-Activity Relationship investigation was carried out.

Cyclopropane-1,2-dicarboxylic acids as new tools for the biophysical investigation of O-acetylserine sulfhydrylases by fluorimetric methods and saturation transfer difference (STD) NMR.

Cysteine is a building block for many biomolecules that are crucial for living organisms. O-Acetylserine sulfhydrylase (OASS), present in bacteria and plants but absent in mammals, catalyzes the last step of cysteine biosynthesis. This enzyme has been deeply investigated because, beside the biosynthesis of cysteine, it exerts a series of "moonlighting" activities in bacteria. We have previously reported a series of molecules capable of inhibiting Salmonella typhimurium (S. typhymurium) OASS isoforms at nanomolar concentrations, using a combination of computational and spectroscopic approaches. The cyclopropane-1,2-dicarboxylic acids presented herein provide further insights into the binding mode of small molecules to OASS enzymes. Saturation transfer difference NMR (STD-NMR) was used to characterize the molecule/enzyme interactions for both OASS-A and B. Most of the compounds induce a several fold increase in fluorescence emission of the pyridoxal 5'-phosphate (PLP) coenzyme upon binding to either OASS-A or OASS-B, making these compounds excellent tools for the development of competition-binding experiments.

Metabolite Profiling and Bioactivities of Leaves, Stems, and Flowers of Rumex usambarensis (Dammer) Dammer, a Traditional African Medicinal Plant.

The comprehensive identification of secondary metabolites represents a fundamental step for the assessment of bioactivities and pharmacological properties of traditional herbal drugs. Rumex usambarensis (Dammer) Dammer has been described as a multipurpose remedy in different African traditional pharmacopoeias, but its phytochemical profile has not been properly investigated. Herein we report a high throughput metabolomic screening, based on ultra-high performance liquid chromatography-travelling wave ion mobility spectrometry quadrupole time-of-flight (UHPLC-TWINS-QTOF), which was performed for the first time on different R. usambarensis plant parts. By applying high-resolution mass spectrometry-based metabolomics and chemometric analysis, a complete discrimination of different aerial parts was obtained, with the annotation of 153 significant metabolites in leaves, stems, and flowers, suggesting an easy authentication and discrimination route. Phytochemical data were correlated to antimicrobial and antioxidant properties. Flavonoids, benzopyranes, chromones, and xanthones derivatives, along with a richer phytocomplex, might be responsible for the stronger bioactivities obtained from flowers.

Expanding the knowledge around antitubercular 5-(2-aminothiazol-4-yl)isoxazole-3-carboxamides: Hit-to-lead optimization and release of a novel antitubercular chemotype via scaffold derivatization.

Tuberculosis is one of the deadliest infectious diseases in the world, and the increased number of multidrug-resistant and extensively drug-resistant strains is a reason for concern. We have previously reported a series of substituted 5-(2-aminothiazol-4-yl)isoxazole-3-carboxamides with growth inhibitory activity against Mycobacterium tuberculosis strains and low propensity to be substrate of efflux pumps. Encouraged by these preliminary results, we have undertaken a medicinal chemistry campaign to determine the metabolic fate of these compounds and to delineate a reliable body of Structure-Activity Relationships. Keeping intact the (thiazol-4-yl)isoxazole-3-carboxamide core, as it is deemed to be the pharmacophore of the molecule, we have extensively explored the structural modifications able to confer good activity and avoid rapid clearance. Also, a small set of analogues based on isostere manipulation of the 2-aminothiazole were prepared and tested, with the aim to disclose novel antitubercular chemotypes. These studies, combined, were instrumental in designing improved compounds such as 42g and 42l, escaping metabolic degradation by human liver microsomes and, at the same time, maintaining good antitubercular activity against both drug-susceptible and drug-resistant strains.

Identification of Human Alanine-Glyoxylate Aminotransferase Ligands as Pharmacological Chaperones for Variants Associated with Primary Hyperoxaluria Type 1.

Primary hyperoxaluria type I (PH1) is a rare kidney disease due to the deficit of alanine:glyoxylate aminotransferase (AGT), a pyridoxal-5'-phosphate-dependent enzyme responsible for liver glyoxylate detoxification, which in turn prevents oxalate formation and precipitation as kidney stones. Many PH1-associated missense mutations cause AGT misfolding. Therefore, the use of pharmacological chaperones (PCs), small molecules that promote correct folding, represents a useful therapeutic option. To identify ligands acting as PCs for AGT, we first performed a small screening of commercially available compounds. We tested each molecule by a dual approach aimed at defining the inhibition potency on purified proteins and the chaperone activity in cells expressing a misfolded variant associated with PH1. We then performed a chemical optimization campaign and tested the resulting synthetic molecules using the same approach. Overall, the results allowed us to identify a promising hit compound for AGT and draw conclusions about the requirements for optimal PC activity.

Inhibitors of O-Acetylserine Sulfhydrylase with a Cyclopropane-Carboxylic Acid Scaffold Are Effective Colistin Adjuvants in Gram Negative Bacteria.

Antibacterial adjuvants are of great significance, since they allow one to downscale the therapeutic dose of conventional antibiotics and reduce the insurgence of antibacterial resistance. Herein, we report that O-acetylserine sulfhydrylase (OASS) inhibitors could be used as colistin adjuvants to treat infections caused by critical pathogens spreading worldwide, Escherichia coli, Salmonella enterica serovar Typhimurium, and Klebsiella pneumoniae. Starting from a hit compound endowed with a nanomolar dissociation constant, we have rationally designed and synthesized a series of derivatives to be tested against S. Typhimurium OASS isoenzymes, StOASS-A and StOASS-B. All acidic derivatives have shown good activities in the nanomolar range against both OASS isoforms in vitro. Minimal Inhibitory Concentrations (MICs) were then evaluated, as well as compounds' toxicity. The compounds endowed with good activity in vitro and low cytotoxicity have been challenged as a potential colistin adjuvant against pathogenic bacteria in vitro and the fractional inhibitory concentration (FIC) index has been calculated to define additive or synergistic effects. Finally, the target engagement inside the S. Typhimurium cells was confirmed by using a mutant strain in which the OASS enzymes were inactivated. Our results provide a robust proof of principle supporting OASS as a potential nonessential antibacterial target to develop a new class of adjuvants.

Discovery of Substituted (2-Aminooxazol-4-yl)Isoxazole-3-carboxylic Acids as Inhibitors of Bacterial Serine Acetyltransferase in the Quest for Novel Potential Antibacterial Adjuvants.

Many bacteria and actinomycetales use L-cysteine biosynthesis to increase their tolerance to antibacterial treatment and establish a long-lasting infection. In turn, this might lead to the onset of antimicrobial resistance that currently represents one of the most menacing threats to public health worldwide. The biosynthetic machinery required to synthesise L-cysteine is absent in mammals; therefore, its exploitation as a drug target is particularly promising. In this article, we report a series of inhibitors of Salmonella thyphimurium serine acetyltransferase (SAT), the enzyme that catalyzes the rate-limiting step of L-cysteine biosynthesis. The development of such inhibitors started with the virtual screening of an in-house library of compounds that led to the selection of seven structurally unrelated hit derivatives. A set of molecules structurally related to hit compound 5, coming either from the original library or from medicinal chemistry efforts, were tested to determine a preliminary structure-activity relationship and, especially, to improve the inhibitory potency of the derivatives, that was indeed ameliorated by several folds compared to hit compound 5 Despite these progresses, at this stage, the most promising compound failed to interfere with bacterial growth when tested on a Gram-negative model organism, anticipating the need for further research efforts.

Investigational Studies on a Hit Compound Cyclopropane-Carboxylic Acid Derivative Targeting O-Acetylserine Sulfhydrylase as a Colistin Adjuvant.

Antibacterial adjuvants are of great significance, since they allow the therapeutic dose of conventional antibiotics to be lowered and reduce the insurgence of antibiotic resistance. Herein, we report that an O-acetylserine sulfhydrylase (OASS) inhibitor can be used as a colistin adjuvant to treat infections caused by Gram-positive and Gram-negative pathogens. A compound that binds OASS with a nM dissociation constant was tested as an adjuvant of colistin against six critical pathogens responsible for infections spreading worldwide, Escherichia coli, Salmonella enterica serovar Typhimurium, Klebisiella pneumoniae, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Staphylococcus pseudintermedius. The compound showed promising synergistic or additive activities against all of them. Knockout experiments confirmed the intracellular target engagement supporting the proposed mechanism of action. Moreover, compound toxicity was evaluated by means of its hemolytic activity against sheep defibrinated blood cells, showing a good safety profile. The 3D structure of the compound in complex with OASS was determined at 1.2 Å resolution by macromolecular crystallography, providing for the first time structural insights about the nature of the interaction between the enzyme and this class of competitive inhibitors. Our results provide a robust proof of principle supporting OASS as a potential nonessential antibacterial target to develop a new class of adjuvants and the structural basis for further structure-activity relationship studies.

Antimicrobial peptides (AMPs): a patent review (2015-2020).

Introduction: Antimicrobial peptides are a large class of compounds that are part of innate immune response found among all classes of life and are considered promising compounds to deal with antimicrobial resistance. These AMPs have been demonstrated to have some advantages over the traditional antibiotics with a broad spectrum of antimicrobial activities and even overcome bacterial drug-resistance. Areas covered: The present review represents a comprehensive analysis of patents and patent applications available on Espacenet, from the year 2015 to 2020 referring to the therapeutic use of AMPs. Expert opinion: There are important examples about the use of antimicrobial peptides in clinical practice (e.g. polimixin b, colistin, etc.). AMPs are usually inspired by nature being produced by different living organisms as defensive and/or competition mechanisms. Despite limitations related to their development in classical drug discovery pipeline, they are endowed with relevant advantages, such as an unlimited reservoir of organisms able to produce new AMPs and they represent good starting point upon which to develop new antimicrobials.

2-Aminooxazole as a Novel Privileged Scaffold in Antitubercular Medicinal Chemistry.

To obtain effective eradication of numerous infectious diseases such as tuberculosis, it is important to supply the medicinal chemistry arsenal with novel chemical agents. Isosterism and bioisosterism are widely known concepts in the field of early drug discovery, and in several cases, rational isosteric replacements have contributed to improved efficacy and physicochemical characteristics throughout the hit-to-lead optimization process. However, sometimes the synthesis of isosteres might not be as straightforward as that of the parent compounds, and therefore, novel synthetic strategies must be elaborated. In this regard, we herein report the evaluation of a series of N-substituted 4-phenyl-2-aminooxazoles that, despite being isosteres of a widely used nucleus such as the 2-aminothiazole, have been only seldom explored. After elaboration of a convenient synthetic strategy, a small set of 2-aminothiazoles and their 2-aminooxazole counterparts were compared with regard to antitubercular activity and physicochemical characteristics.

Inhibition of Nonessential Bacterial Targets: Discovery of a Novel Serine O-Acetyltransferase Inhibitor.

In ϒ-proteobacteria and Actinomycetales, cysteine biosynthetic enzymes are indispensable during persistence and become dispensable during growth or acute infection. The biosynthetic machinery required to convert inorganic sulfur into cysteine is absent in mammals; therefore, it is a suitable drug target. We searched for inhibitors of Salmonella serine acetyltransferase (SAT), the enzyme that catalyzes the rate-limiting step of l-cysteine biosynthesis. The virtual screening of three ChemDiv focused libraries containing 91 243 compounds was performed to identify potential SAT inhibitors. Scaffold similarity and the analysis of the overall physicochemical properties allowed the selection of 73 compounds that were purchased and evaluated on the recombinant enzyme. Six compounds displaying an IC50 <100 µM were identified via an indirect assay using Ellman's reagent and then tested on a Gram-negative model organism, with one of them being able to interfere with bacterial growth via SAT inhibition.

Sodium Hyaluronate Nanocomposite Respirable Microparticles to Tackle Antibiotic Resistance with Potential Application in Treatment of Mycobacterial Pulmonary Infections.

Tuberculosis resistant cases have been estimated to grow every year. Besides Mycobacterium tuberculosis, other mycobacterial species are responsible for an increasing number of difficult-to-treat infections. To increase efficacy of pulmonary treatment of mycobacterial infections an inhalable antibiotic powder targeting infected alveolar macrophages (AMs) and including an efflux pump inhibitor was developed. Low molecular weight sodium hyaluronate sub-micron particles were efficiently loaded with rifampicin, isoniazid and verapamil, and transformed in highly respirable microparticles (mean volume diameter: 1 µm) by spray drying. These particles were able to regenerate their original size upon contact with aqueous environment with mechanical stirring or sonication. The in vitro drugs release profile from the powder was characterized by a slow release rate, favorable to maintain a high drug level inside AMs. In vitro antimicrobial activity and ex vivo macrophage infection assays employing susceptible and drug resistant strains were carried out. No significant differences were observed when the powder, which did not compromise the AMs viability after a five-day exposure, was compared to the same formulation without verapamil. However, both preparations achieved more than 80% reduction in bacterial viability irrespective of the drug resistance profile. This approach can be considered appropriate to treat mycobacterial respiratory infections, regardless the level of drug resistance.

The Aryl Hydrocarbon Receptor (AhR) Mediates the Counter-Regulatory Effects of Pelargonidins in Models of Inflammation and Metabolic Dysfunctions.

Pelargonidins are anthocyanidins thought to be beneficial for the human health, although controversies exist over the doses needed and the unclear mechanism of action, along with poor systemic bioavailability. One putative target of pelargonidins is the aryl hydrocarbon receptor (AhR). A synthetic pelargonidin (Mt-P) was synthesized by the methylation of the pelargonidin (the natural compound indicated as P). Mt-P transactivated the AhR with an EC50 of 1.97 µM and was ~2-fold more potent than the natural compound. In vitro Mt-P attenuated pro-inflammatory activities of Raw264.7 macrophage cells in an AhR-dependent manner. In vivo, administration of the Mt-P in Balb/c mice resulted in a dose-dependent attenuation of signs and symptoms of colitis induced by TNBS. A dose of 5 mg/kg Mt-P, but not the natural compound P, reversed intestinal inflammation and increased expression of Tnf-α, Ifn-Æ´, and Il-6, while promoted the expansion of regulatory T cells and M2 macrophages. In C57BL/6J mice fed a high fat diet (HFD), Mt-P attenuated body weight gain, intestinal and liver inflammation, and ameliorated insulin sensitivity, while worsened liver steatosis by up-regulating the liver expression of Cd36 and Apo100b. These effects were abrogated by AhR gene ablation. Mt-P is a synthetic pelargonidin endowed with robust AhR agonist activity that exerts beneficial effects in murine models of inflammation and metabolic dysfunction.

Biochemical Characterization of Aspergillus fumigatus AroH, a Putative Aromatic Amino Acid Aminotransferase.

The rise in the frequency of nosocomial infections is becoming a major problem for public health, in particular in immunocompromised patients. Aspergillus fumigatus is an opportunistic fungus normally present in the environment directly responsible for lethal invasive infections. Recent results suggest that the metabolic pathways related to amino acid metabolism can regulate the fungus-host interaction and that an important role is played by enzymes involved in the catabolism of L-tryptophan. In particular, in A. fumigatus L-tryptophan regulates Aro genes. Among them, AroH encodes a putative pyridoxal 5'-phosphate-dependent aminotransferase. Here we analyzed the biochemical features of recombinant purified AroH by spectroscopic and kinetic analyses corroborated by in silico studies. We found that the protein is dimeric and tightly binds the coenzyme forming a deprotonated internal aldimine in equilibrium with a protonated ketoenamine form. By setting up a new rapid assay method, we measured the kinetic parameters for the overall transamination of substrates and we demonstrated that AroH behaves as an aromatic amino acid aminotransferase, but also accepts L-kynurenine and α-aminoadipate as amino donors. Interestingly, computational approaches showed that the predicted overall fold and active site topology of the protein are similar to those of its yeast ortholog, albeit with some differences in the regions at the entrance of the active site, which could possibly influence substrate specificity. Should targeting fungal metabolic adaptation be of therapeutic value, the results of the present study may pave the way to the design of specific AroH modulators as potential novel agents at the host/fungus interface.