Rapid and Accurate Prediction and Scoring of Water Molecules in Protein Binding Sites

Water plays a critical role in ligand-protein interactions. However, it is still challenging to predict accurately not only where water molecules prefer to bind, but also which of those water molecules might be displaceable. The latter is often seen as a route to optimizing affinity of potential drug candidates. Using a protocol we call WaterDock, we show that the freely available AutoDock Vina tool can be used to predict accurately the binding sites of water molecules. WaterDock was validated using data from X-ray crystallography, neutron diffraction and molecular dynamics simulations and correctly predicted 97% of the water molecules in the test set. In addition, we combined data-mining, heuristic and machine learning techniques to develop probabilistic water molecule classifiers. When applied to WaterDock predictions in the Astex Diverse Set of protein ligand complexes, we could identify whether a water molecule was conserved or displaced to an accuracy of 75%. A second model predicted whether water molecules were displaced by polar groups or by non-polar groups to an accuracy of 80%. These results should prove useful for anyone wishing to undertake rational design of new compounds where the displacement of water molecules is being considered as a route to improved affinity

Arginase Flavonoid Anti-Leishmanial in Silico Inhibitors Flagged against Anti-Targets

Arginase, a drug target for the treatment of leishmaniasis, is involved in the biosynthesis of polyamines. Flavonoids are interesting natural compounds found in many foods and some of them may inhibit this enzyme. The MetIDB database containing 5667 compounds was screened using an EIIP/AQVN filter and 3D QSAR to find the most promising candidate compounds. In addition, these top hits were screened in silico versus human arginase and an anti-target battery consisting of cytochromes P450 2a6, 2c9, 3a4, sulfotransferase, and the pregnane-X-receptor in order to flag their possible interactions with these proteins involved in the metabolism of substances. The resulting compounds may have promise to be further developed for the treatment of leishmaniasis

A Fragmenting Protocol with Explicit Hydration for Calculation of Binding Enthalpies of Target-Ligand Complexes at a Quantum Mechanical Level

Optimization of the enthalpy component of binding thermodynamics of drug candidates is a successful pathway of rational molecular design. However, the large size and missing hydration structure of target-ligand complexes often hinder such optimizations with quantum mechanical (QM) methods. At the same time, QM calculations are often necessitated for proper handling of electronic effects. To overcome the above problems, and help the QM design of new drugs, a protocol is introduced for atomic level determination of hydration structure and extraction of structures of target-ligand complex interfaces. The protocol is a combination of a previously published program MobyWat, an engine for assigning explicit water positions, and Fragmenter, a new tool for optimal fragmentation of protein targets. The protocol fostered a series of fast calculations of ligand binding enthalpies at the semi-empirical QM level. Ligands of diverse chemistry ranging from small aromatic compounds up to a large peptide helix of a molecular weight of 3000 targeting a leukemia protein were selected for systematic investigations. Comparison of various combinations of implicit and explicit water models demonstrated that the presence of accurately predicted explicit water molecules in the complex interface considerably improved the agreement with experimental results. A single scaling factor was derived for conversion of QM reaction heats into binding enthalpy values. The factor links molecular structure with binding thermodynamics via QM calculations. The new protocol and scaling factor will help automated optimization of binding enthalpy in future molecular design projects

In silico selection and in vitro testing of natural and synthetic compounds inhibiting Leishmania spp. growth

Predmet naučnog istraživanja ove doktorske disertacije ,,In silico selekcija i in vitro ispitivanje prirodnih i sintetskih jedinjenja sa potencijalnom inhibitornom aktivnošću na rast Leishmania spp." su prirodna i sintetska jedinjenja sa potencijalnim inhibitornim dejstvom na protozoe iz roda lajšmanija. Osnovni zadatak studije je bio da se in silico metodologijom pronađu odgovarajući kandidati koji pripadaju takvoj grupi jedinjenja i da se njihova aktivnost potvrdi in vitro eksperimentima. Dodatni zadatak je bilo postavljanje hipoteze o mehanizmu dejstva inhibitora na ciljna mesta u telu parazita na osnovu razumevanja interakcija hemijskih jedinjenja kandidata i odabranih metaboličkih enzima. Stečena saznanja imaju za cilj da doprinesu razvoj i dizajn novih aktivnih jedinjenja koja imaju potencijalnu hemoterapijsku primenu u lečenju lajšmanioze sa minimalnim toksičnim dejstvom na ćelije domaćina. Prva faza istraživanja obuhvata: (1) Definisanje kriterijuma za virtuelni skrining baza hemijskih jedinjenja upotrebom molekulskih deskriptora: elektron-jon interakcionog potencijala – EIIP i prosečnog kvazivalentnog broja – AQVN, 3D modela odnosa strukture i aktivnosti – 3D-QSAR i farmakofornog 3D modela zasnovanog na strukturi liganda; (2) Primena homolognog modelovanja enzima čija struktura nije kristalografski utvrđena; (3) razvoj kombinovanog protokola za virtuelni skrining zasnovanog na strukturi liganda i enzma u cilju dobijanja novih inhibitora rasta lajšmanije; (4) In silico skrining anti-targeta, tj. ispitivanje interakcija potencijalnih inhibitora sa enzimima koji učestvuju u njihovom metabolizmu i koji su prisutni u ćelijama domaćina; (5) Predviđanje apsorpcije, distribucije, metabolizma, ekskrecije i toksičnosti – ADMET i fizičkohemijskih osobina kandidata. Druga faza istraživanja obuhvata: (1) Odabir kandidata sa potencijalnom aktivnošću na lajšmanije na osnovu in silico skrininga, a koji se mogu pronaći u katalogu dobavljača ili koji se pronalaze u dostupnoj bazi prirodnih proizvoda; (2) Dobijanje odabranih kandidata automatizovanom sintezom; (3) In vitro ispitivanje aktivnosti inhibitora porekla na Leishmania spp. Definisan je protokol virtuelnog skrininga, a potom primenjen za pretragu baze MetIDB od 5.667 jedinjenja, upotrebom EIIP/AQVN filtera i 3D-QSAR. Jedinjenja sa najboljim rezultatima pretrage su potom dokovana u model humane i arginaze lajšmanije kao i anti-target modele koje čine citohromi P450 familije enzima 2a6, 2c9, 3a4, sulfotransferaza i pregnan-X-receptor sa ciljem označavanja neželjenih receptor-ligand interakcija u toku metabolizma. Upotreba ove metode utiče na donošenje odluke o odabiru kandidata koji potencijalno imaju umanjena toksična i neželjena dejstva u toku lečenja lajšmanioze. Pripremljena je serija novih sintetskih jedinjenja, derivata oksadiazola i indolizina. Za ova jedinjenja su izračunati EIIP i AQVN deskriptori na osnovu kojih je formiran domen za upotrebu u narednom koraku, tj. skriningu zasnovanom na strukturi liganda. Najbolji kandidati su dokovani u model humane arginaze i finalno okarakterisani na osnovu dokinga u grupe anti-target enzima, kako bi se ispitala mogućnost interakcije sa proteinima esencijalnim za njihov metabolizam. Osam kandidata je testirano in vitro. Rezultati pokazuju da antilajšmanijalna aktivnost postoji za tri od osam kandidata. Najbolji kandidat sa 2,18 μM IC50 na amastigote Leishmania donovani u makrofagama, predstavlja interesantnu strukturu za razvoj novih agensa sa dejstvom na lajšmanije. Primenom strukturnih podataka izozima, modelovana je 3D struktura Leishmania infantum alternativne NADH dehidrogenaze (tip 2) upotrebom metoda prepoznavanja homologa. Virtuelnim skriningom je izvršena pretraga potencijalnih inhibitora koji kao ciljno mesto dejstva imaju LiNDH-2 ubikvinon-vezivno mesto...he subject of this dissertation titled: "In silico selection and in vitro testing of natural and synthetic compounds inhibitors of Leishmania spp growth" is the natural and synthetic compounds that act as growth inhibitors of the protozoa of the genus Leishmania. The main objective of this study is to identify such compounds using in silico methodology and evaluate the activity by in vitro experiments. A further aim is to hypothesize the mechanism of action against specific targets within the parasite body, based on an understanding of molecular interactions with specific metabolic enzymes. These research findings should be used to develop targeted drugs against leishmaniasis that reduce the risk of toxic effects on host cells. The first phase consists of (1) defining the criteria for virtual screening of compound databases using the descriptors: electron-ion interaction potential – EIIP and average quasivalence number - AQVN, quantitative structure-activity relationship - 3D-QSAR and ligand-based pharmacophore model. (2) The application of homology modeling to elucidate target structure necessary for receptor-ligand interactions with parasite and the host, for those enzymes for which crystallographic structure is not available; (3) The development of VS protocol comprising ligand-based and structure-based methods to obtain inhibitors of Leishmania growth; (4) In silico anti-target screening, viz.i.e. qualification of receptor-ligand interactions of the potential inhibitors against host metabolic enzymes; (5) The prediction of absorption, distribution, metabolism, excretion and toxicity - ADMET and physicochemical properties of the selected candidates. The second phase of research involves: (1) the selection of candidates with potential activity against leishmaniasis based on in silico screening, found in the supplier's catalog or in the available database of natural products; (2) the extraction of selected candidates by automated synthesis; (3) the in vitro study of the inhibitory activity of Leishmania spp. After defining VS protocol, an initial screening was performed using an EIIP/AQVN filter and 3D-QSAR against the MetIDB database of 5,667 compounds. Top hits were screened in silico against human and Leishmania arginase and an anti-target model consisting of cytochromes P450 2a6, 2c9, 3a4, sulfotransferase, and the pregnane X receptor to flag unfavorable ligand-protein interactions during compound metabolism. Using this method as a filter affects the decision of choosing compounds which may produce fewer toxic and adverse effects in the treatment of leishmaniasis. A series of novel oxadiazoles and indolizine-containing compounds were synthesized. Then, EIIP and AQVN values were calculated for each compound and only those that belonged to a predefined domain during ligand-based virtual screening were selected. Molecular docking of the selected candidates using a parasite arginase model was performed. The top hits were further docked to human arginase and characterized by docking to anti-target enzymes to mark their possible interactions with enzymes essential for their metabolism. Eight candidate compounds were selected for further experimental testing. The results show measurable in vitro anti-leishmanial activity for three out of eight compounds..

In silico selection and in vitro testing of natural and synthetic compounds inhibiting Leishmania spp. growth

Predmet naučnog istraživanja ove doktorske disertacije ,,In silico selekcija i in vitro ispitivanjeprirodnih i sintetskih jedinjenja sa potencijalnom inhibitornom aktivnošću na rast Leishmania spp." suprirodna i sintetska jedinjenja sa potencijalnim inhibitornim dejstvom na protozoe iz roda lajšmanija.Osnovni zadatak studije je bio da se in silico metodologijom pronađu odgovarajući kandidati koji pripadajutakvoj grupi jedinjenja i da se njihova aktivnost potvrdi in vitro eksperimentima. Dodatni zadatak je bilopostavljanje hipoteze o mehanizmu dejstva inhibitora na ciljna mesta u telu parazita na osnovurazumevanja interakcija hemijskih jedinjenja kandidata i odabranih metaboličkih enzima. Stečena saznanjaimaju za cilj da doprinesu razvoj i dizajn novih aktivnih jedinjenja koja imaju potencijalnu hemoterapijskuprimenu u lečenju lajšmanioze sa minimalnim toksičnim dejstvom na ćelije domaćina.Prva faza istraživanja obuhvata: (1) Definisanje kriterijuma za virtuelni skrining baza hemijskihjedinjenja upotrebom molekulskih deskriptora: elektron-jon interakcionog potencijala – EIIP i prosečnogkvazivalentnog broja – AQVN, 3D modela odnosa strukture i aktivnosti – 3D-QSAR i farmakofornog3D modela zasnovanog na strukturi liganda; (2) Primena homolognog modelovanja enzima čija strukturanije kristalografski utvrđena; (3) razvoj kombinovanog protokola za virtuelni skrining zasnovanog nastrukturi liganda i enzma u cilju dobijanja novih inhibitora rasta lajšmanije; (4) In silico skrining anti-targeta,tj. ispitivanje interakcija potencijalnih inhibitora sa enzimima koji učestvuju u njihovom metabolizmu ikoji su prisutni u ćelijama domaćina; (5) Predviđanje apsorpcije, distribucije, metabolizma, ekskrecije itoksičnosti – ADMET i fizičkohemijskih osobina kandidata.Druga faza istraživanja obuhvata: (1) Odabir kandidata sa potencijalnom aktivnošću na lajšmanijena osnovu in silico skrininga, a koji se mogu pronaći u katalogu dobavljača ili koji se pronalaze u dostupnojbazi prirodnih proizvoda; (2) Dobijanje odabranih kandidata automatizovanom sintezom; (3) In vitroispitivanje aktivnosti inhibitora porekla na Leishmania spp.Definisan je protokol virtuelnog skrininga, a potom primenjen za pretragu baze MetIDB od 5.667jedinjenja, upotrebom EIIP/AQVN filtera i 3D-QSAR. Jedinjenja sa najboljim rezultatima pretrage supotom dokovana u model humane i arginaze lajšmanije kao i anti-target modele koje čine citohromi P450familije enzima 2a6, 2c9, 3a4, sulfotransferaza i pregnan-X-receptor sa ciljem označavanja neželjenihreceptor-ligand interakcija u toku metabolizma. Upotreba ove metode utiče na donošenje odluke oodabiru kandidata koji potencijalno imaju umanjena toksična i neželjena dejstva u toku lečenjalajšmanioze.Pripremljena je serija novih sintetskih jedinjenja, derivata oksadiazola i indolizina. Za ovajedinjenja su izračunati EIIP i AQVN deskriptori na osnovu kojih je formiran domen za upotrebu unarednom koraku, tj. skriningu zasnovanom na strukturi liganda. Najbolji kandidati su dokovani u modelhumane arginaze i finalno okarakterisani na osnovu dokinga u grupe anti-target enzima, kako bi se ispitalamogućnost interakcije sa proteinima esencijalnim za njihov metabolizam. Osam kandidata je testirano invitro. Rezultati pokazuju da antilajšmanijalna aktivnost postoji za tri od osam kandidata. Najbolji kandidatsa 2,18 μM IC50 na amastigote Leishmania donovani u makrofagama, predstavlja interesantnu strukturu zarazvoj novih agensa sa dejstvom na lajšmanije.Primenom strukturnih podataka izozima, modelovana je 3D struktura Leishmania infantumalternativne NADH dehidrogenaze (tip 2) upotrebom metoda prepoznavanja homologa. Virtuelnimskriningom je izvršena pretraga potencijalnih inhibitora koji kao ciljno mesto dejstva imaju LiNDH-2ubikvinon-vezivno mesto...The subject of this dissertation titled: "In silico selection and in vitro testing of natural and syntheticcompounds inhibitors of Leishmania spp growth" is the natural and synthetic compounds that act asgrowth inhibitors of the protozoa of the genus Leishmania. The main objective of this study is to identifysuch compounds using in silico methodology and evaluate the activity by in vitro experiments. A furtheraim is to hypothesize the mechanism of action against specific targets within the parasite body, based onan understanding of molecular interactions with specific metabolic enzymes. These research findingsshould be used to develop targeted drugs against leishmaniasis that reduce the risk of toxic effects onhost cells.The first phase consists of (1) defining the criteria for virtual screening of compound databasesusing the descriptors: electron-ion interaction potential – EIIP and average quasivalence number - AQVN,quantitative structure-activity relationship - 3D-QSAR and ligand-based pharmacophore model. (2) Theapplication of homology modeling to elucidate target structure necessary for receptor-ligand interactionswith parasite and the host, for those enzymes for which crystallographic structure is not available; (3)The development of VS protocol comprising ligand-based and structure-based methods to obtaininhibitors of Leishmania growth; (4) In silico anti-target screening, viz.i.e. qualification of receptor-ligandinteractions of the potential inhibitors against host metabolic enzymes; (5) The prediction of absorption,distribution, metabolism, excretion and toxicity - ADMET and physicochemical properties of theselected candidates.The second phase of research involves: (1) the selection of candidates with potential activityagainst leishmaniasis based on in silico screening, found in the supplier's catalog or in the available databaseof natural products; (2) the extraction of selected candidates by automated synthesis; (3) the in vitro studyof the inhibitory activity of Leishmania spp.After defining VS protocol, an initial screening was performed using an EIIP/AQVN filter and3D-QSAR against the MetIDB database of 5,667 compounds. Top hits were screened in silico againsthuman and Leishmania arginase and an anti-target model consisting of cytochromes P450 2a6, 2c9, 3a4,sulfotransferase, and the pregnane X receptor to flag unfavorable ligand-protein interactions duringcompound metabolism. Using this method as a filter affects the decision of choosing compounds whichmay produce fewer toxic and adverse effects in the treatment of leishmaniasis.A series of novel oxadiazoles and indolizine-containing compounds were synthesized. Then,EIIP and AQVN values were calculated for each compound and only those that belonged to a predefineddomain during ligand-based virtual screening were selected. Molecular docking of the selected candidatesusing a parasite arginase model was performed. The top hits were further docked to human arginase andcharacterized by docking to anti-target enzymes to mark their possible interactions with enzymes essentialfor their metabolism. Eight candidate compounds were selected for further experimental testing. Theresults show measurable in vitro anti-leishmanial activity for three out of eight compounds..

Water in Protein Cavities: Free Energy, Entropy, Enthalpy, and its Influences on Protein Structure and Flexibility

Complexes of the antibiotics novobiocin and clorobiocin with DNA gyrase are illustrative of the importance of bound water to binding thermodynamics. Mutants resistantto novobiocin as well as those with a decreased affinity for novobiocin over clorobiocinboth involve a less favorable entropy of binding, which more than compensates for amore favorable enthalpy, and additional water molecules at the proteinligandinterface.Free energy, enthalpy, and entropy for these water molecules were calculated by thermodynamicintegration computer simulations. The calculations show that addition of thewater molecules is entropically unfavorable, with values that are comparable to the measuredentropy differences. The free energies and entropies correlate with the change inthe number of hydrogen bonds due to the addition of water molecules.To examine the wide variety of cavities available to water molecules inside proteins,a model of the protein cavities is developed with the local environment treated at atomicdetail and the nonlocal environment treated approximately. The cavities are then changedto vary in size and in the number of hydrogen bonds available to a water molecule insidethe cavity. The free energy, entropy, and enthalpy change for the transfer of a watermolecule to the cavity from the bulk liquid is calculated from thermodynamic integration.The results of the model are close to those of similar cavities calculated using the fullprotein and solvent environment. As the number of hydrogen bonds resulting from theaddition of the water molecule increases, the free energy decreases, as the enthalpic gainof making a hydrogen bond outweighs the entropic cost. Changing the volume of thecavity has a smaller effect on the thermodynamics. Once the hydrogen bond contributionis taken into account, the volume dependence on free energy, entropy, and enthalpy issmall and roughly the same for a hydrophobic cavity as a hydrophilic cavity.The influences of bound water on protein structure and influences are also evaluatedby performing molecular dynamics simulation for proteins with and without boundwater. Four proteins are simulated, the wildtypebovine pancreatic trypsin inhibitor(BPTI), the wildtypehen egg white lysozyme (HEWL), and two variants of the wildtypeStaphylococcal nuclease (SNase), PHS and PHS/V66E. The simulation reveals that allthese four proteins suffer structural changes upon the removing of bound water molecules,as indicating by their increased RMSD values with respect to the crystal structures. Threeout of the four proteins, BPTI, HEWL, and the PHS mutant of SNase have increased flexibility,while no apparent flexibility change is seen in the PHS/V66E variant of SNase

Water in Protein Cavities: Free Energy, Entropy, Enthalpy, and its Influences on Protein Structure and Flexibility

Complexes of the antibiotics novobiocin and clorobiocin with DNA gyrase are illustrative of the importance of bound water to binding thermodynamics. Mutants resistantto novobiocin as well as those with a decreased affinity for novobiocin over clorobiocinboth involve a less favorable entropy of binding, which more than compensates for amore favorable enthalpy, and additional water molecules at the proteinligandinterface.Free energy, enthalpy, and entropy for these water molecules were calculated by thermodynamicintegration computer simulations. The calculations show that addition of thewater molecules is entropically unfavorable, with values that are comparable to the measuredentropy differences. The free energies and entropies correlate with the change inthe number of hydrogen bonds due to the addition of water molecules.To examine the wide variety of cavities available to water molecules inside proteins,a model of the protein cavities is developed with the local environment treated at atomicdetail and the nonlocal environment treated approximately. The cavities are then changedto vary in size and in the number of hydrogen bonds available to a water molecule insidethe cavity. The free energy, entropy, and enthalpy change for the transfer of a watermolecule to the cavity from the bulk liquid is calculated from thermodynamic integration.The results of the model are close to those of similar cavities calculated using the fullprotein and solvent environment. As the number of hydrogen bonds resulting from theaddition of the water molecule increases, the free energy decreases, as the enthalpic gainof making a hydrogen bond outweighs the entropic cost. Changing the volume of thecavity has a smaller effect on the thermodynamics. Once the hydrogen bond contributionis taken into account, the volume dependence on free energy, entropy, and enthalpy issmall and roughly the same for a hydrophobic cavity as a hydrophilic cavity.The influences of bound water on protein structure and influences are also evaluatedby performing molecular dynamics simulation for proteins with and without boundwater. Four proteins are simulated, the wildtypebovine pancreatic trypsin inhibitor(BPTI), the wildtypehen egg white lysozyme (HEWL), and two variants of the wildtypeStaphylococcal nuclease (SNase), PHS and PHS/V66E. The simulation reveals that allthese four proteins suffer structural changes upon the removing of bound water molecules,as indicating by their increased RMSD values with respect to the crystal structures. Threeout of the four proteins, BPTI, HEWL, and the PHS mutant of SNase have increased flexibility,while no apparent flexibility change is seen in the PHS/V66E variant of SNase

Including Tightly-Bound Water Molecules in de Novo Drug Design. Exemplification through the in Silico Generation of Poly(ADP-ribose)polymerase Ligands

Different strategies for the in silico generation of ligand molecules in the binding site of poly(ADP-ribose)-polymerase (PARP) were studied in order to observe the effect of the targeting and displacement of tightly bound water molecules. Several molecular scaffolds were identified as having better interactions in the binding site when targeting one or two tightly bound water molecules in the NAD binding site. Energy calculations were conducted in order to assess the ligand-protein and ligand-water-protein interactions of different functional groups of the generated ligands. These calculations were used to evaluate the energetic consequences of the presence of tightly bound water molecules and to identify those that contribute favorably to the binding of ligands