Biochemical Studies on Human Ornithine Aminotransferase Support a Cell-Based Enzyme Replacement Therapy in the Gyrate Atrophy of the Choroid and Retina.

The gyrate atrophy of the choroid and retina (GACR) is a rare genetic disease for which no definitive cure is available. GACR is due to the deficit of ornithine aminotransferase (hOAT), a pyridoxal 5'-phosphate-dependent enzyme responsible for ornithine catabolism. The hallmark of the disease is plasmatic ornithine accumulation, which damages retinal epithelium leading to progressive vision loss and blindness within the fifth decade. Here, we characterized the biochemical properties of tetrameric and dimeric hOAT and evaluated hOAT loaded in red blood cells (RBCs) as a possible enzyme replacement therapy (ERT) for GACR. Our results show that (i) hOAT has a relatively wide specificity for amino acceptors, with pyruvate being the most suitable candidate for ornithine catabolism within RBCs; (ii) both the tetrameric and dimeric enzyme can be loaded in RBC retaining their activity; and (iii) hOAT displays reduced stability in plasma, but is partly protected from inactivation upon incubation in a mixture mimicking the intracellular erythrocyte environment. Preliminary ex vivo experiments indicate that hOAT-loaded RBCs are able to metabolize extracellular ornithine at a concentration mimicking that found in patients, both in buffer and, although with lower efficiency, in plasma. Overall, our data provide a proof of concept that an RBC-mediated ERT is feasible and can be exploited as a new therapeutic approach in GACR.

Estimating the hydrophobicity extent of molecular fragments using reversed-phase liquid chromatography.

A fast HPLC method was developed to study the hydrophobicity extent of pharmaceutically relevant molecular fragments. By this strategy, the reduced amount of sample available for physico-chemical evaluations in early-phase drug discovery programs does not represent a limiting factor. The sixteen acid fragments investigated were previously synthesized also determining potentiometrically their experimental log D values. For four fragments it was not possible to determine such property since their values were outside of the instrumental working range (2 < pKa  < 12). An RP-HPLC method was therefore optimized. For each scrutinized method, some derived chromatographic indices were calculated, and Pearson's correlation coefficient (r) allowed to select the so-called "φ0 index" as the best correlating with the log D. The w s p H ${}_w/pH$ was fixed at 3.5 and a modification of some variables [organic modifier (methanol vs. ACN), stationary phase (octyl vs. octadecyl), presence/absence of the additives n-octanol, n-butylamine, and n-octylamine], allowed to select the best correlation conditions, producing a r = 0.94 (p < 0.001). Importantly, the φ0 index enabled the estimation of log D values for four fragments which were unattainable by potentiometric titration. Moreover, a series of molecular descriptors were calculated to identify the chemical characteristics of the fragments explaining the obtained φ0 . The number of hydrogen bond donors and the index of cohesive interaction correlated with the experimental data.

Multi-Gram Scale Synthesis and Characterization of Mometasone Furoate EP Impurity C.

Mometasone furoate is a synthetic corticosteroid used in the treatment of skin inflammatory conditions, hay fever and asthma. The industrial manufacturing routes to mometasone furoate are generally accompanied by the formation of numerous process impurities that need to be detected and quantified, as requested by regulatory authorities. The ready availability of such impurities in the required quantity and purity is therefore essential for toxicological studies, analytical method development and process validation. Herein, we report the multi-gram scale preparation of 21'-chloro-(16'α-methyl-3',11',20'-trioxo-pregna-1',4'-dien-17'-yl)-furan-2-carboxylate (mometasone furoate EP impurity C), one of the known impurities of mometasone furoate. This study also includes the systematic investigation of the final acylation step, as well as the characterization of the difuroate enol ether intermediate and its conversion to the target impurity C.

Critical Assessment of a Structure-Based Screening Campaign for IDO1 Inhibitors: Tips and Pitfalls.

Over the last two decades, indoleamine 2,3-dioxygenase 1 (IDO1) has attracted wide interest as a key player in immune regulation, fostering the design and development of small molecule inhibitors to restore immune response in tumor immunity. In this framework, biochemical, structural, and pharmacological studies have unveiled peculiar structural plasticity of IDO1, with different conformations and functional states that are coupled to fine regulation of its catalytic activity and non-enzymic functions. The large plasticity of IDO1 may affect its ligand recognition process, generating bias in structure-based drug design campaigns. In this work, we report a screening campaign of a fragment library of compounds, grounding on the use of three distinct conformations of IDO1 that recapitulate its structural plasticity to some extent. Results are instrumental to discuss tips and pitfalls that, due to the large plasticity of the enzyme, may influence the identification of novel and differentiated chemical scaffolds of IDO1 ligands in structure-based screening campaigns.

Fragment based drug design and diversity-oriented synthesis of carboxylic acid isosteres.

The medicinal chemist toolbox is plenty of (bio)isosteres when looking for a carboxylic acid replacement. However, systematic assessment of acid surrogates is often time consuming and expensive, while prediction of both physicochemical properties (logP and logD) as well as acidity would be desirable at early discovery stages for a better analog design. Herein in this work, to enable decision making on a project, we have synthesized by employing a Diversity-Oriented Synthetic (DOS) methodology, a small library of molecular fragments endowed with acidic properties. By combining in-silico and experimental methodologies these compounds were chemically characterized and, particularly, with the aim to know their physicochemical properties, the aqueous ionization constants (pKa), partition coefficients logD and logP of each fragment was firstly estimated by using molecular modeling studies and then validated by experimental determinations. A face to face comparison between data and the corresponding carboxylic acid might help medicinal chemists in finding the best replacement to be used. Finally, in the framework of Fragment Based Drug Design (FBDD) the small library of fragments obtained with our approach showed good versatility both in synthetic and physico-chemical properties.

Enantioselective HPLC Analysis to Assist the Chemical Exploration of Chiral Imidazolines.

In the present work, we illustrate the ability of high-performance liquid chromatography (HPLC) analysis to assist the synthesis of chiral imidazolines within our medicinal chemistry programs. In particular, a Chiralpak® IB® column containing cellulose tris(3,5-dimethylphenylcarbamate) immobilized onto a 5 µm silica gel was used for the enantioselective HPLC analysis of chiral imidazolines synthesized in the frame of hit-to-lead explorations and designed for exploring the effect of diverse amide substitutions. Very profitably, reversed-phase (RP) conditions succeeded in resolving the enantiomers in nine out of the 10 investigated enantiomeric pairs, with α values always higher than 1.10 and RS values up to 2.31. All compounds were analysed with 50% (v) water while varying the content of the two organic modifiers acetonitrile and methanol. All the employed eluent systems were buffered with 40 mM ammonium acetate while the apparent pH was fixed at 7.5. Based on the experimental results, the prominent role of π-π stacking interactions between the substituted electron-rich phenyl groups outside of the polymeric selector and the complementary aromatic region in defining analyte retention and stereodiscrimination was identified. The importance of compound polarity in explaining the retention behaviour with the employed RP system was readily evident when a quantitative structure-property relationship study was performed on the retention factor values (k) of the 10 compounds, as computed with a 30% (v) methanol containing mobile phase. Indeed, good Pearson correlation coefficients of retention factors (r - log k1st = -0.93; r - log k2nd = -0.94) were obtained with a water solubility descriptor (Ali-logS). Interestingly, a n-hexane/chloroform/ethanol (88:10:2, v/v/v)-based non-standard mobile phase allowed the almost base-line enantioseparation (α = 1.06; RS = 1.26) of the unique compound undiscriminated under RP conditions.

Chiral separation of helical chromenes with chloromethyl phenylcarbamate polysaccharide-based stationary phases.

Two chloromethyl phenylcarbamate-based chiral stationary phases, one containing an amylose-type chiral selector (Lux Amylose 2, from Phenomenex) and the other a cellulose-type one (Lux Cellulose-4, from Phenomenex), were successfully used for the chiral resolution of three helical chromenes featuring a helicene-like structure. The compound bearing a phenyl substituent on the helicene-like structure was enantioresolved at 25°C with Lux Cellulose-4 and a n-hexane/1-propanol 99:1 v/v eluent. With a n-hexane/2-propanol 99.8:0.2 v/v mobile phase, the same column (operated at 35°C) provided the separation of the four isomers of the compound having a hexyl residue on the helicene-like motif and an additional asymmetric carbon. Lux Amylose-2 was necessary for the enantioseparation of the compound having the sole hexyl residue on the helical scaffold. For the last compound a n-hexane/2-propanol 99.8:0.2 v/v eluent was used, and the column temperature was fixed at 5°C. The enantiomer elution order was appraised by using electronic circular dichroism and theoretical calculations. Notably, different thermodynamics of retention and enantioseparation were observed for molecules with pronounced structural similarity, that is, the enantiomer pairs of the compound containing the additional asymmetric carbon atom. Indeed, both entropically and enthalpically controlled adsorption and separation processes were observed.

Hydrophobic Amino Acid Content in Onions as Potential Fingerprints of Geographical Origin: The Case of Rossa da Inverno sel. Rojo Duro.

In this study, we were interested in comparing the amino acid profile in a specific variety of onion, Rossa da inverno sel. Rojo Duro, produced in two different Italian sites: the Cannara (Umbria region) and Imola (Emilia Romagna region) sites. Onions were cultivated in a comparable manner, mostly in terms of the mineral fertilization, seeding, and harvesting stages, as well as good weed control. Furthermore, in both regions, the plants were irrigated by the water sprinkler method and subjected to similar temperature and weather conditions. A further group of Cannara onions that were grown by micro-irrigation was also evaluated. After the extraction of the free amino acid mixture, an ion-pairing reversed-phase (IP-RP) HPLC method allowed for the separation and the evaporative light scattering detection of almost all the standard proteinogenic amino acids. However, only the peaks corresponding to leucine (Leu), phenylalanine (Phe), and tryptophan (Trp), were present in all the investigated samples and they were unaffected from the matrix interfering peaks. The use of the beeswarm/box plots revealed that the content of Leu and Phe were markedly influenced by the geographical origin of the onions (with *** p.

Investigating the allosteric reverse signalling of PARP inhibitors with microsecond molecular dynamic simulations and fluorescence anisotropy.

The inhibition of the poly(ADP-ribose) polymerase (PARP) family members is a strategy pursued for the development of novel therapeutic agents in a range of diseases, including stroke, cardiac ischemia, cancer, inflammation and diabetes. Even though some PARP-1 inhibitors have advanced to clinical setting for cancer therapy, a great deal of attention is being devoted to understand the polypharmacology of current PARP inhibitors. Besides blocking the catalytic activity, recent works have shown that some PARP inhibitors exhibit a poisoning activity, by trapping the enzyme at damaged sites of DNA and forming cytotoxic complexes. In this study we have used microsecond molecular dynamics to study the allosteric reverse signalling that is at the basis of such an effect. We show that Olaparib, but not Veliparib and HYDAMTIQ, is able to induce a specific conformational drift of the WGR domain of PARP-1, which stabilizes PARP-1/DNA complex through the locking of several salt bridge interactions. Fluorescence anisotropy assays support such a mechanism, providing the first experimental evidence that HYDAMTIQ, a potent PARP inhibitor with neuroprotective properties, is less potent than Olaparib to trap PARP-1/DNA complex.

Sustainable Protocols for Cellulose Nanocrystals Synthesis from Tomato Waste and Their Antimicrobial Properties against Pseudomonas syringae pv. tomato.

Nanotechnology is rapidly gaining ground in crop protection, with the growing quest for sustainable nanopesticides and nanocarriers for plant pathogen management. Among them, cellulose nanocrystals (CNC) are emerging as innovative agrofood-waste-derived antimicrobial materials. In this work, new chemical and enzymatic CNC extraction methods from tomato harvest residues were evaluated. The obtained nanomaterials were characterized and tested for their antimicrobial properties on Pseudomonas syringae pv. tomato (Pto), the causal agent of bacterial speck disease on tomato. Both protocols were efficient. The enzymatic extraction method was greener, producing purer CNC at slightly lower yield. The obtained CNC, although they weakly inhibited cell growth and did not promote reactive oxygen species (ROS) formation, provoked bacterial aggregation and the inhibition of biofilm production and swimming motility. Both protocols produced CNC with similar morpho-chemical features, as well as promising antimicrobial activity against plant bacterial pathogens, suggesting their potential role in sustainable crop protection strategies. The new protocols could be a valuable alternative to conventional methods.

Boosting Immunity and Management against Wheat Fusarium Diseases by a Sustainable, Circular Nanostructured Delivery Platform.

Fusarium head blight (FHB) and Fusarium crown rot (FCR) are managed by the application of imidazole fungicides, which will be strictly limited by 2030, as stated by the European Green Deal. Here, a novel and eco-sustainable nanostructured particle formulation (NPF) is presented by following the principles of the circular economy. Cellulose nanocrystals (CNC) and resistant starch were obtained from the bran of a high amylose (HA) bread wheat and employed as carrier and excipient, while chitosan and gallic acid were functionalized as antifungal and elicitor active principles. The NPF inhibited conidia germination and mycelium growth, and mechanically interacted with conidia. The NPF optimally reduced FHB and FCR symptoms in susceptible bread wheat genotypes while being biocompatible on plants. The expression level of 21 genes involved in the induction of innate immunity was investigated in Sumai3 (FHB resistant) Cadenza (susceptible) and Cadenza SBEIIa (a mutant characterized by high-amylose starch content) and most of them were up-regulated in Cadenza SBEIIa spikes treated with the NPF, indicating that this genotype may possess an interesting genomic background particularly responsive to elicitor-like molecules. Quantification of fungal biomass revealed that the NPF controlled FHB spread, while Cadenza SBEIIa was resistant to FCR fungal spread. The present research work highlights that the NPF is a powerful weapon for FHB sustainable management, while the genome of Cadenza SBEIIa should be investigated deeply as particularly responsive to elicitor-like molecules and resistant to FCR fungal spread.

Dual species sphingosine-1-phosphate lyase inhibitors to combine antifungal and anti-inflammatory activities in cystic fibrosis: a feasibility study.

Cystic fibrosis (CF) is an autosomal recessive disorder characterized by respiratory failure due to a vicious cycle of defective Cystic Fibrosis Transmembrane conductance Regulator (CFTR) function, chronic inflammation and recurrent bacterial and fungal infections. Although the recent introduction of CFTR correctors/potentiators has revolutionized the clinical management of CF patients, resurgence of inflammation and persistence of pathogens still posit a major concern and should be targeted contextually. On the background of a network-based selectivity that allows to target the same enzyme in the host and microbes with different outcomes, we focused on sphingosine-1-phosphate (S1P) lyase (SPL) of the sphingolipid metabolism as a potential candidate to uniquely induce anti-inflammatory and antifungal activities in CF. As a feasibility study, herein we show that interfering with S1P metabolism improved the immune response in a murine model of CF with aspergillosis while preventing germination of Aspergillus fumigatus conidia. In addition, in an early drug discovery process, we purified human and A. fumigatus SPL, characterized their biochemical and structural properties, and performed an in silico screening to identify potential dual species SPL inhibitors. We identified two hits behaving as competitive inhibitors of pathogen and host SPL, thus paving the way for hit-to-lead and translational studies for the development of drug candidates capable of restraining fungal growth and increasing antifungal resistance.

Fast chromatographic determination of the bile salt critical micellar concentration.

In a line of research focused on the design, synthesis and development of new bile acid-based compounds, the physico-chemical profile of the molecules must be thoroughly explored and analyzed. In this scenario, a fast and reliable information on the critical micellar concentration (CMC) of specific compounds through a profitable chromatographic parameter can be of aid to rationally direct the synthesis of new molecular entities, mainly during the early stages of the drug-discovery process. The derived 'chromatographic hydrophobicity index' (CHI), usually employed for a fast access to the log P/log D value of physico-chemically diverse compounds and obtained via RP-gradient elution, was for the first time engaged in the bile acid field. Accordingly, 14 unconjugated bile acids harboured with a different number, position and orientation of hydroxy groups, as well as other substituents onto the steroidal backbone and side chain, were selected to build up a calibration curve. Such a collection of compounds was rationally assembled in order to manage an almost continuous range of CMC values (spanning the spectrophotometrically obtained CMCs between 5 and 25 mM). A high degree of correlation between CMC and CHI values was obtained (R(2) and cross-validated R(xv)(2) of the pCMC vs CHI plot equal to 0.975 and 0.966, respectively). A selected new subset of five confidential research bile acids with experimental CMCs in the range 6-19 mM was finally recruited to validate the proposed method. The high statistical quality of the established mathematical model turned out into a very appreciable predictive power.

Recent advances in urea- and thiourea-containing compounds: focus on innovative approaches in medicinal chemistry and organic synthesis.

Urea and thiourea represent privileged structures in medicinal chemistry. Indeed, these moieties constitute a common framework of a variety of drugs and bioactive compounds endowed with a broad range of therapeutic and pharmacological properties. Herein, we provide an overview of the state-of-the-art of urea and thiourea-containing pharmaceuticals. We also review the diverse approaches pursued for (thio)urea bioisosteric replacements in medicinal chemistry applications. Finally, representative examples of recent advances in the synthesis of urea- and thiourea-based compounds by enabling chemical tools are discussed.

One Key and Multiple Locks: Substrate Binding in Structures of Tryptophan Dioxygenases and Hydroxylases.

Since its discovery at the beginning of the past century, the essential nutrient l-Tryptophan (l-Trp) and its catabolic pathways have acquired an increasing interest in an ever wider scientific community for their pivotal roles in underlying many important physiological functions and associated pathological conditions. As a consequence, enzymes catalyzing rate limiting steps along l-Trp catabolic pathways - including IDO1, TDO, TPH1 and TPH2 - have turned to be interesting drug targets for the design and development of novel therapeutic agents for different disorders such as carcinoid syndrome, cancer and autoimmune diseases. This article provides a fresh comparative overview on the most recent advancements that crystallographic studies, biophysical and computational works have brought on structural aspects and molecular recognition patterns of these enzymes toward l-Trp. Finally, a conformational analysis of l-Trp is also discussed as part of the molecular recognition process governing the binding of a substrate to its cognate enzymes.

A Green Nanostructured Pesticide to Control Tomato Bacterial Speck Disease.

Bacterial speck disease, caused by Pseudomonas syringae pv. tomato (Pst), is one of the most pervasive biological adversities in tomato cultivation, in both industrial and in table varieties. In this work synthesis, biochemical and antibacterial properties of a novel organic nanostructured pesticide composed of chitosan hydrochloride (CH) as active ingredient, cellulose nanocrystals (CNC) as nanocarriers and starch as excipient were evaluated. In order to study the possibility of delivering CH, the effects of two different types of starches, extracted from a high amylose bread wheat (high amylose starch-HA Starch) and from a control genotype (standard starch-St Starch), were investigated. Nanostructured microparticles (NMP) were obtained through the spray-drying technique, revealing a CH loading capacity proximal to 50%, with a CH release of 30% for CH-CNC-St Starch NMP and 50% for CH-CNC-HA Starch NMP after 24 h. Both NMP were able to inhibit bacterial growth in vitro when used at 1% w/v. Moreover, no negative effects on vegetative growth were recorded when NMP were foliar applied on tomato plants. Proposed nanostructured pesticides showed the capability of diminishing Pst epiphytical survival during time, decreasing disease incidence and severity (from 45% to 49%), with results comparable to one of the most used cupric salt (hydroxide), pointing out the potential use of CH-CNC-Starch NMP as a sustainable and innovative ally in Pst control strategies.

Adenosine deaminase - A target for new piperazine derivatives.

The level of adenosine deaminase (ADA) activity increases in pathological effusions. Therefore, the concentration of its substrate, anti-inflammatory adenosine, decreases, thereby aggravating inflammation. Hence, the quest for ADA inhibiting compounds is an actual problem in medicine and pharmacology. This work describes the inhibition of bovine ADA by new synthesized piperazine compounds. 15 compounds were screened; IC50 values for 5 more potent ones of them were between 3.4 and 98.6 µg/ml. The inhibition of activity of intracellular and ecto- forms of ADA by the most effective "compound 1" was of competitive nature. For these two forms of enzyme, the inhibition constants, Ki (1.5 and 115 µM) and IC50 values (6.5 and 480 µM), respectively, differed by nearly two orders. The constant of bimolecular interaction KSV between "compound 1" and the tryptophan residues in ADA was estimated in fluorescence quenching study as of 0.145 ± 0.027 µM. Finally, the molecular interactions between "compound 1" and the bovine enzyme ADA were highlighted through molecular docking studies.

Chiral ligand-exchange separation and resolution of extremely rigid glutamate analogs: 1-aminospiro[2.2]pentyl-1,4-dicarboxylic acids.

Owing to their chelation ability, a series of fully constrained L-Glu analogs formed by the spiro-union of two cyclopropane rings (1-aminospiro[2.2]pentyl-1,4-dicarboxylic acids, ASPED A-D), was submitted to chiral ligand-exchange chromatographic (CLEC) analysis. As the initial step, two methodologically different chiral devices were evaluated. A chiral stationary phase (CSP) obtained by dynamic coating of C(18) chains with the S-trityl-(R)-cysteine ((R)-STC) was used first with this objective. The lack of separation of the enantiomers of ASPED C and D prompted us to utilize the chiral mobile phase (CMP) prepared from O-benzyl-(S)-serine ((S)-OBS). The latter afforded complete separation of the four pairs of enantiomers. For all the pairs, quantum mechanical investigations shed light on the main features responsible for the different enantiomer recognition mechanism with (S)-OBS. The validated analytical method was then fruitfully adopted for semi-preparative-scale isolation of the enantiomers of ASPED C.

New Insights from Crystallographic Data: Diversity of Structural Motifs and Molecular Recognition Properties between Groups of IDO1 Structures.

A large number of crystallographic structures of IDO1 in different ligand-bound and -unbound states have been disclosed over the last decade. Yet, only a few of them have been exploited for structure-based drug design (SBDD) campaigns. In this study, we analyzed the structural motifs and molecular-recognition properties of three groups of IDO1 structures: 1) structures containing the heme group and inhibitors in the catalytic site; 2) heme-free structures of IDO1; 3) substrate-bound structures of IDO1. The results suggest that unrelated conformations of the enzyme have been solved with different ligand-induced changes of secondary motifs that localize even in regions remote from the catalytic site. Moreover, the study identified an uncharted region of molecular-recognition space covered by IDO1 binding sites that could guide the selection of diverse structures for additional SBDD studies aimed at the identification of novel lead compounds with differentiated chemical scaffolds.

Quantitative analysis of cucurbitane-type triterpenes in Ibervillea sonorae extracts: Relationship study with their antiproliferative activity.

Ibervillea sonorae (Cucurbitaceae) is a Mexican plant commonly used by local population for its hypoglycaemic activity. Root extracts showed also other different biological activities, including antimicrobial, antifungal, antioxidant and anti-inflammatory activity. Main components of this plant are cucurbitacins, steroid-like triterpenes that possess, among others, antiproliferative activity. In previous studies, kinoin A and cucurbitacin IIb extracted from I. sonorae showed antiproliferative and apoptotic effects against different cancer cell lines. Based on all the above, a RP-HPLC method was developed and validated for the quantitative analysis of these two compounds in I. sonorae root extracts obtained with different extraction conditions. In the present study, the quantitative analysis of kinoin B diglycoside in all the extracts was performed as well. As a result, no direct correlation was found between the antiproliferative activity (IC50) against human cervical cancer cell line (HeLa) and the composition of the above three compounds. Only a slight statically significant negative correlation was observed between IC50s and the content of kinoin A (r = 0.29, p = 0.12), meaning that, at least in part, this is the main compound among the three, contributing to the antiproliferative activity on the real samples. Accordingly, a synergistic effect by the phytocomplex components can account for the observed antiproliferative activity of the methanolic extracts towards HeLa cells.