Rapid detection of soil carbonates by means of NIR spectroscopy, deep learning methods and phase quantification by powder Xray diffraction

Soil NIR spectral absorbance/reflectance libraries are utilized towards improving agricultural production and analysis of soil properties which are key prerequisite for agroecological balance and environmental sustainability. Carbonates in particular, represent a soil property which is mostly affected even by mild, let alone extreme, changes of environmental conditions during climate change. In this study we propose a rapid and efficient way to predict carbonates content in soil by means of FT NIR reflectance spectroscopy and by use of deep learning methods. We exploited multiple machine learning methods, such as: 1) a MLP Regressor and 2) a CNN and compare their performance with other traditional ML algorithms such as PLSR, Cubist and SVM on the combined dataset of two NIR spectral libraries: KSSL (USDA), a dataset of soil samples reflectance spectra collected nationwide, and LUCAS TopSoil (European Soil Library) which contains soil sample absorbance spectra from all over the European Union, and use them to predict carbonate content on never before seen soil samples. Soil samples in KSSL and in TopSoil spectral libraries were acquired in the spectral region of visNIR, however in this study, only the NIR spectral region was utilized. Quantification of carbonates by means of Xray Diffraction is in good agreement with the volumetric method and the MLP prediction. Our work contributes to rapid carbonates content prediction in soil samples in cases where: 1) no volumetric method is available and 2) only NIR spectra absorbance data are available. Up till now and to the best of our knowledge, there exists no other study, that presents a prediction model trained on such an extensive dataset with such promising results on unseen data, undoubtedly supporting the notion that deep learning models present excellent prediction tools for soil carbonates content.Comment: 39 pages, 5 figure

The Interaction of the Chemotherapeutic Drug Chlorambucil with Human Glutathione Transferase A1-1: Kinetic and Structural Analysis

Glutathione transferases (GSTs) are enzymes that contribute to cellular detoxification by catalysing the nucleophilic attack of glutathione (GSH) on the electrophilic centre of a number of xenobiotic compounds, including several chemotherapeutic drugs. In the present work we investigated the interaction of the chemotherapeutic drug chlorambucil (CBL) with human GSTA1-1 (hGSTA1-1) using kinetic analysis, protein crystallography and molecular dynamics. In the presence of GSH, CBL behaves as an efficient substrate for hGSTA1-1. The rate-limiting step of the catalytic reaction between CBL and GSH is viscosity-dependent and kinetic data suggest that product release is rate-limiting. The crystal structure of the hGSTA1-1/ CBL-GSH complex was solved at 2.1 A° resolution by molecular replacement. CBL is bound at the H-site attached to the thiol group of GSH, is partially ordered and exposed to the solvent, making specific interactions with the enzyme. Molecular dynamics simulations based on the crystal structure indicated high mobility of the CBL moiety and stabilization of the Cterminal helix due to the presence of the adduct. In the absence of GSH, CBL is shown to be an alkylating irreversible inhibitor for hGSTA1-1. Inactivation of the enzyme by CBL followed a biphasic pseudo-first-order saturation kinetics with approximately 1 mol of CBL per mol of dimeric enzyme being incorporated. Structural analysis suggested that the modifying residue is Cys112 which is located at the entrance of the H-site. The results are indicative of a structural communication between the subunits on the basis of mutually exclusive modification of Cys112, indicating that the two enzyme active sites are presumably coordinated

Crystallographic study of bioactive Ruthenium complexes with model proteins

The Ru coordination compounds attract particular interest as alternative anticancer metallodrugs due to their lower toxicity and higher specificity against specific cancer lines compared to the widely used coordination compounds of Pt. Their pharmacological profile has been ascribed to their interactions with proteins, as several previous studies have provided evidence that DNA is not the primary target. In this regard, the investigation of their binding with target proteins, by a detailed structural analysis of the binding mode geometrical features and interactions which reveal the specificity for the protein binding sites, is crucial for the understanding of their action and the design of novel coordination compounds of Ru with optimal pharmacological properties. In the present thesis, high-resolution X-ray crystal structures of complexes of Ru(II) and Ru(III) coordination compounds with protein targets are presented and extensively analyzed. The examined compounds were the “half sandwich”-type Ru(II) coordination compound [RuII(1,4,7-trithiacyclononane)(ethane-1,2-diamine)Cl]+ (RuTE) and the antimetastatic metallodrug imidazolium trans-[tetrachloride(S-dimethyl sufoxide) (1H-imidazole) ruthenate(III)] (NAMI-A, a widely known promising metallodrug of Ru(III) already in Phase I/II clinical trials). The protein targets were two enzymes: the Hen Egg White Lysozyme (HEWL) and the Proteinase K (PK). HEWL is an ordinary model system of protein metalation studies and it was used as a target for both RuTE and NAMI-A, whereas PK was used here for the first time as such, forming a complex with RuTE. The determination of high (atomic) resolution crystal structures by X-ray crystallography and their analysis revealed the following:RuTE is coordinated by a covalent bond to the solvent exposed residues of Aspartic acid of the enzyme-targets (Asp101 in HEWL, Asp200 and Asp260 in PK), followed by a release of a chloride ligand while retaining the two chelating ligands of the compound. The crystal structures show for the first time the binding of a “half-sandwich” type compound of Ru(II) to a protein, that maintains its coordination sphere almost intact. From the structural analysis, several significant conclusions were derived considering the binding mode of RuTE with protein residues in specific binding sites and the stabilization of the complexes via hydrogen bonds, hydrophobic and aromatic interactions. The complex formed between NAMI-A and HEWL was studied by analyzing a series of near atomic-resolution X-ray crystal structures determined by crystals obtained at four time points of soaking HEWL crystals with excess NAMI-A (1.5, 8.0, 26 and 98 h). These structures elucidate a series of binding events starting from the noncovalent interaction of intact NAMI-A ions with HEWL (1.5 h), followed by the stepwise exchange of all Ru ligands except for 1H-imidazole (26 h) to the final “ruthenated” protein comprising one aquated Ru ion coordinated to histidine-15 of HEWL (98 h). The structural data clearly support a two-step mechanism of protein ruthenation, illustrating the ligand-mediated recognition step of the process.Οι ενώσεις συναρμογής του Ru ελκύουν ιδιαίτερο ενδιαφέρον ως εναλλακτικά αντικαρκινικά μεταλλοφάρμακα καθώς εμφανίζουν χαμηλότερη τοξικότητα και υψηλότερη εκλεκτικότητα έναντι συγκεκριμένων καρκινικών σειρών σε σχέση με τις ευρέως χρησιμοποιούμενες ενώσεις συναρμογής του Pt. Προηγούμενες μελέτες, εστιασμένες στη σύνθεση και βιολογική αξιολόγηση μεταλλοφαρμάκων Ru, έχουν αποδώσει τις ευνοϊκές ιδιότητες τους και το φαρμακολογικό τους προφίλ κυρίως στις αλληλεπιδράσεις τους με πρωτεΐνες καθώς το DNA δεν αποτελεί τον βασικό στόχο τους. Ως εκ τούτου, η διερεύνηση ερωτημάτων σχετικά με τον τρόπο πρόσδεσης, τις αλληλεπιδράσεις και την εκλεκτικότητα τους σε πρωτεϊνικές περιοχές πρόσδεσης αποτελούν κλειδιά για τη κατανόηση της δράσης τους ανοίγοντας νέους δρόμους στον σχεδιασμό ενώσεων συναρμογής του Ru με βέλτιστες φαρμακολογικές ιδιότητες. Στην παρούσα διατριβή τα ερωτήματα αυτά διερευνήθηκαν με δομική μελέτη, μέσω κρυσταλλογραφίας ακτίνων-Χ, συμπλόκων μεταξύ ενώσεων συναρμογής Ru(II)/Ru(III) και πρωτεϊνικών στόχων. Χρησιμοποιήθηκαν οι ακόλουθες ενώσεις συναρμογής: Η Ένωση Συναρμογής-1 (ΕΣ-1) [RuII(1,4,7-trithiacyclononane) (ethane-1,2-diamine)Cl]+, η οποία αποτελεί ένωση συναρμογής τύπου «half-sandwich» του Ru(II) και η Ένωση Συναρμογής-2 (ΕΣ-2) [ImidazoleH]trans-[RuCl4(dmso-S)(imidazole)], η ευρέως γνωστή ως ΝΑΜΙ-Α ένωση συναρμογής του Ru(III), πολλά υποσχόμενο μεταλλοφάρμακο του Ru που έχει περάσει ήδη στις Φάσεις Ι/ΙΙ κλινικών δοκιμών. Ως πρωτεϊνικοί στόχοι για την ΕΣ-1 επιλέχθηκαν δύο ένζυμα: η Λυσοζύμη (hen egg-white lysozyme, HEWL), η οποία αποτελεί σύνηθες μοντέλο για τη διερεύνηση της πρόσδεσης μεταλλοφαρμάκων σε αυτήν, και η πρωτεϊνάση Κ (PK), η οποία χρησιμοποιήθηκε για πρώτη φορά ως μοντέλο πρωτεΐνης σε μελέτες μετάλλωσης, αναδεικνύοντας έναν πολύ καλό οδηγό για παρόμοιες έρευνες. Για την ΕΣ-2, η μελέτη έγινε με στόχο αποκλειστικά τη λυσοζύμη. Ο προσδιορισμός των κρυσταλλικών δομών επετεύχθη σε υψηλή (ατομική) διακριτότητα και από την ανάλυσή τους προέκυψαν τα ακόλουθα αποτελέσματα και συμπεράσματα. Η ΕΣ-1 (RuTE) συναρμόζεται μέσω ενός ομοιοπολικού δεσμού με τα εκτεθειμένα στο διαλύτη αμινοξικά κατάλοιπα ασπαρτικού οξέος των ενζύμων-στόχων (Asp101 στην HEWL, Asp200 και Asp260 στην PΚ), έπειτα από απελευθέρωση του χλωρίου υποκαταστάτη ενώ παραμένουν οι δύο χηλικοί υποκαταστάτες της ένωσης. Οι κρυσταλλογραφικά προσδιορισμένες δομές παρουσιάζουν για πρώτη φορά την πρόσδεση σε πρωτεΐνη μίας ένωσης τύπου «half sandwich» του Ru(II) με τη σφαίρα συναρμογής της σχεδόν ακέραια. Από τη δομική ανάλυση προκύπτουν σημαντικά συμπεράσματα για τις περιοχές και τον τρόπο πρόσδεσης του RuTE σε συγκεκριμένα αμινοξικά κατάλοιπα των πρωτεϊνών και για τον ρόλο των υποκαταστατών του Ru(II) τόσο στην εκλεκτικότητα του RuTE σε σχέση με το ηλεκτροστατικό δυναμικό των ενζύμων-στόχων όσο και στην σταθεροποίηση των συμπλόκων μέσω δεσμών υδρογόνου, υδροφοβικών και αρωματικών αλληλεπιδράσεων. Η ΕΣ-2 (ΝΑΜΙ-Α) μελετήθηκε στο σύμπλοκό της με λυσοζύμη, μέσω της ανάλυσης 4 δομών που προσδιορίστηκαν από κρυστάλλους λυσοζύμης εμβαπτισμένους σε ΝΑΜΙ-Α για διαφορετικά χρονικά διαστήματα και συγκεκριμένα για 1.5, 8.0, 26 και 98 ώρες αντίδρασης. Οι 3 πρώτες δομές αποκαλύπτουν για πρώτη φορά την αρχική, μη ομοιοπολική, αλληλεπίδραση ακέραιου ΝΑΜΙ-Α με τη λυσοζύμη, ακολουθούμενη από μία σταδιακή ανταλλαγή όλων των υποκαταστατών του Ru εκτός του ιμιδαζολίου μέχρι τις 26 ώρες. Στα σύμπλοκα αυτά, εμφανίζονται 3 θέσεις πρόσδεσης του ΝΑΜΙ-Α στη HEWL, με το ιμιδαζόλιο να καθορίζει τη σταθεροποίηση τους. Στην τελική ένωση προσθήκης του Ru στις 98 ώρες, ένα ιόν Ru βρίσκεται συναρμοσμένο στην ιστιδίνη-15 της λυσοζύμης. Η δομή του συμπλόκου στις 98 ώρες βρίσκεται σε συμφωνία με προηγούμενες όμοιες κρυσταλλογραφικές μελέτες. Τα αποτελέσματα αυτά υποστηρίζουν έναν μηχανισμό δύο βημάτων στην πρωτεϊνική ρουθηνίωση και φανερώνουν τον ρόλο των υποκαταστατών στο βήμα της αναγνώρισης κατά τη διαδικασία αυτή

Insights into the Protein Ruthenation Mechanism by Antimetastatic Metallodrugs: High-Resolution X-ray Structures of the Adduct Formed between Hen Egg-White Lysozyme and NAMI-A at Various Time Points

The pharmacological profile of medicinally relevant Ru(III) coordination compounds has been ascribed to their interactions with proteins, as several studies have provided evidence that DNA is not the primary target. In this regard, numerous spectroscopic and crystallographic studies have indicated that the Ru(III) ligands play an important role in determining the metal binding site, acting as the recognition element in the early stages of the protein–complex formation. Herein, we present a series of near-atomic-resolution X-ray crystal structures of the adducts formed between the antimetastatic metallodrug imidazolium trans-[tetrachlorido(S-dimethyl sufoxide)(1H-imidazole)ruthenate(III)] (NAMI-A) and hen egg-white lysozyme (HEWL). These structures elucidate a series of binding events starting from the noncovalent interaction of intact NAMI-A ions with HEWL (1.5 h), followed by the stepwise exchange of all Ru ligands except for 1H-imidazole (26 h) to the final “ruthenated” protein comprising one aquated Ru ion coordinated to histidine-15 of HEWL (98 h). Our structural data clearly support a two-step mechanism of protein ruthenation, illustrating the ligand-mediated recognition step of the process

Discovery of Selective Inhibitor Leads by Targeting an Allosteric Site in Insulin-Regulated Aminopeptidase

Insulin-Regulated aminopeptidase (IRAP) is a zinc-dependent aminopeptidase with several important biological functions and is an emerging pharmaceutical target for cognitive enhancement and immune system regulation. Aiming to discover lead-like IRAP inhibitors with enhanced selectivity versus homologous enzymes, we targeted an allosteric site at the C-terminal domain pocket of IRAP. We compiled a library of 2.5 million commercially available compounds from the ZINC database, and performed molecular docking at the target pocket of IRAP and the corresponding pocket of the homologous endoplasmic reticulum aminopeptidase 1 (ERAP1). Of the top compounds that showed high selectivity, 305 were further analyzed by molecular dynamics simulations and free energy calculations, leading to the selection of 33 compounds for in vitro evaluation. Two orthogonal functional assays were employed: one using a small fluorogenic substrate and one following the degradation of oxytocin, a natural peptidic substrate of IRAP. In vitro evaluation suggested that several of the compounds tested can inhibit IRAP, but the inhibition profile was dependent on substrate size, consistent with the allosteric nature of the targeted site. Overall, our results describe several novel leads as IRAP inhibitors and suggest that the C-terminal domain pocket of IRAP is a promising target for developing highly selective IRAP inhibitors

High-resolution crystal structures of a “half sandwich”-type Ru(II) coordination compound bound to hen egg-white lysozyme and proteinase K

The high-resolution X-ray crystal structures of the adducts formed between the “half sandwich”-type Ru(II) coordination compound [Ru$^{II}$(1,4,7-trithiacyclononane)(ethane-1,2-diamine)Cl]$^+$ and two proteins, namely hen egg-white lysozyme and proteinase K, are presented. The structures unveil that upon reaction with both enzymes the Ru(II) compound is coordinated by solvent-exposed aspartate residues after releasing the chloride ligand (Asp101 in lysozyme, Asp200 and Asp260 in proteinase K), while retaining the two chelating ligands. The adduct with Asp101 residue at the catalytic cleft of lysozyme is accompanied by residue-specific conformational changes to accommodate the Ru(II) fragment, whereas the complexes bound at the two calcium-binding sites of proteinase K revealed minimal structural perturbation of the enzyme. To the best of our knowledge, proteinase K is used here for the first time as a model system of protein metalation and these are the first X-ray crystal structures of protein adducts of a Ru(II) coordination compound that maintains its coordination sphere almost intact upon binding. Our data demonstrate the role of ligands in stabilizing the protein adducts via hydrophobic/aromatic or hydrogen-bonding interactions, as well as their underlying role in the selection of specific sites on the electrostatic potential surface of the enzymes

Structural representation illustrating the lock-and-key motif in hGSTA1-1 and a possible mode of communication between the two subunits.

<p>The key residues Met51, Phe52, Cys112 and the GSH–CBL adducts are shown in licorice representation.</p

Superimposed crystallographic structures of hGSTA1-1 (PDB ID: 4HJ2, polypeptide ribbon and carbon atoms in cyan) and hGSTP1-1 (PDB ID: 3CSH, polypeptide ribbon and carbon atoms in orange) complexes with the GSH–CBL adduct.

<p>The interacting Phe10/Phe8 residue of hGST A1-1/P1-1 is shown for comparison.</p

Root mean square fluctuations (RMSF) of hGSTA1-1 Cα atoms calculated for the apo (black) and the holo complex with GSH–CBL (red) from 10-ns molecular dynamics simulations.

<p>The graph shows the mean RMSF value of the two monomers.</p