Controlling Supramolecular Structures of Drugs by Light.

Controlling physicochemical properties of light-unresponsive drugs, by light, prima facie, a paradox approach. We expanded light control by ion pairing light-unresponsive salicylate or ibuprofen to photoswitchable azobenzene counterions, thereby reversibly controlling supramolecular structures, hence the drugs' physicochemical and kinetic properties. The resulting ion pairs photoliquefied into room-temperature ionic liquids under ultraviolet light. Aqueous solutions showed trans-cis-dependent supramolecular structures under a light with wormlike aggregates decomposing into small micelles and vice versa. Light control allowed for permeation through membranes of cis-ibuprofen ion pairs within 12 h in contrast to the trans ion pairs requiring 72 h. In conclusion, azobenzene ion-pairing expands light control of physicochemical and kinetic properties to otherwise light-unresponsive drugs.

Conformational flexibility of DENV NS2B/NS3pro: from the inhibitor effect to the serotype influence.

The dengue virus (DENV) has four well-known serotypes, namely DENV1 to DENV4, which together cause 50-100 million infections worldwide each year. DENV NS2B/NS3pro is a protease recognized as a valid target for DENV antiviral drug discovery. However, NS2B/NS3pro conformational flexibility, involving in particular the NS2B region, is not yet completely understood and, hence, a big challenge for any virtual screening (VS) campaign. Molecular dynamics (MD) simulations were performed in this study to explore the DENV3 NS2B/NS3pro binding-site flexibility and obtain guidelines for further VS studies. MD simulations were done with and without the Bz-nKRR-H inhibitor, showing that the NS2B region stays close to the NS3pro core even in the ligand-free structure. Binding-site conformational states obtained from the simulations were clustered and further analysed using GRID/PCA, identifying four conformations of potential importance for VS studies. A virtual screening applied to a set of 31 peptide-based DENV NS2B/NS3pro inhibitors, taken from literature, illustrated that selective alternative pharmacophore models can be constructed based on conformations derived from MD simulations. For the first time, the NS2B/NS3pro binding-site flexibility was evaluated for all DENV serotypes using homology models followed by MD simulations. Interestingly, the number of NS2B/NS3pro conformational states differed depending on the serotype. Binding-site differences could be identified that may be crucial to subsequent VS studies.

Development, synthesis and structure-activity-relationships of inhibitors of the macrophage infectivity potentiator (Mip) proteins of Legionella pneumophila and Burkholderia pseudomallei.

The bacteria Burkholderia pseudomallei and Legionella pneumophila cause severe diseases like melioidosis and Legionnaire's disease with high mortality rates despite antibiotic treatment. Due to increasing antibiotic resistances against these and other Gram-negative bacteria, alternative therapeutical strategies are in urgent demand. As a virulence factor, the macrophage infectivity potentiator (Mip) protein constitutes an attractive target. The Mip proteins of B. pseudomallei and L. pneumophila exhibit peptidyl-prolyl cis/trans isomerase (PPIase) activity and belong to the PPIase superfamily. In previous studies, the pipecolic acid moiety proved to be a valuable scaffold for inhibiting this PPIase activity. Thus, a library of pipecolic acid derivatives was established guided by structural information and computational analyses of the binding site and possible binding modes. Stability and toxicity considerations were taken into account in iterative extensions of the library. Synthesis and evaluation of the compounds in PPIase assays resulted in highly active inhibitors. The activities can be interpreted in terms of a common binding mode obtained by docking calculations.

An ordered water channel in Staphylococcus aureus FabI: unraveling the mechanism of substrate recognition and reduction.

One third of all drugs in clinical use owe their pharmacological activity to the functional inhibition of enzymes, highlighting the importance of enzymatic targets for drug development. Because of the close relationship between inhibition and catalysis, understanding the recognition and turnover of enzymatic substrates is essential for rational drug design. Although the Staphylococcus aureus enoyl-acyl carrier protein reductase (saFabI) involved in bacterial fatty acid biosynthesis constitutes a very promising target for the development of novel, urgently needed anti-staphylococcal agents, the substrate binding mode and catalytic mechanism remained unclear for this enzyme. Using a combined crystallographic, kinetic, and computational approach, we have explored the chemical properties of the saFabI binding cavity, obtaining a consistent mechanistic model for substrate binding and turnover. We identified a water-molecule network linking the active site with a water basin inside the homo-tetrameric protein, which seems to be crucial for the closure of the flexible substrate binding loop as well as for an effective hydride and proton transfer during catalysis. On the basis of our results, we also derive a new model for the FabI-ACP complex that reveals how the ACP-bound acyl-substrate is injected into the FabI binding crevice. These findings support the future development of novel FabI inhibitors that target the FabI-ACP interface leading to the disruption of the interaction between these two proteins.

Rational design of broad spectrum antibacterial activity based on a clinically relevant enoyl-acyl carrier protein (ACP) reductase inhibitor.

Determining the molecular basis for target selectivity is of particular importance in drug discovery. The ideal antibiotic should be active against a broad spectrum of pathogenic organisms with a minimal effect on human targets. CG400549, a Staphylococcus-specific 2-pyridone compound that inhibits the enoyl-acyl carrier protein reductase (FabI), has recently been shown to possess human efficacy for the treatment of methicillin-resistant Staphylococcus aureus infections, which constitute a serious threat to human health. In this study, we solved the structures of three different FabI homologues in complex with several pyridone inhibitors, including CG400549. Based on these structures, we rationalize the 65-fold reduced affinity of CG400549 toward Escherichia coli versus S. aureus FabI and implement concepts to improve the spectrum of antibacterial activity. The identification of different conformational states along the reaction coordinate of the enzymatic hydride transfer provides an elegant visual depiction of the relationship between catalysis and inhibition, which facilitates rational inhibitor design. Ultimately, we developed the novel 4-pyridone-based FabI inhibitor PT166 that retained favorable pharmacokinetics and efficacy in a mouse model of S. aureus infection with extended activity against Gram-negative and mycobacterial organisms.

Anti-trypanosomal activities and structural chemical properties of selected compound classes.

Potent compounds do not necessarily make the best drugs in the market. Consequently, with the aim to describe tools that may be fundamental for refining the screening of candidates for animal and preclinical studies and further development, molecules of different structural classes synthesized within the frame of a broad screening platform were evaluated for their trypanocidal activities, cytotoxicities against murine macrophages J774.1 and selectivity indices, as well as for their ligand efficiencies and structural chemical properties. To advance into their modes of action, we also describe the morphological and ultrastructural changes exerted by selected members of each compound class on the parasite Trypanosoma brucei. Our data suggest that the potential organelles targeted are either the flagellar pocket (compound 77, N-Arylpyridinium salt; 15, amino acid derivative with piperazine moieties), the endoplasmic reticulum membrane systems (37, bisquaternary bisnaphthalimide; 77, N-Arylpyridinium salt; 68, piperidine derivative), or mitochondria and kinetoplasts (88, N-Arylpyridinium salt; 68, piperidine derivative). Amino acid derivatives with fumaric acid and piperazine moieties (4, 15) weakly inhibiting cysteine proteases seem to preferentially target acidic compartments. Our results suggest that ligand efficiency indices may be helpful to learn about the relationship between potency and chemical characteristics of the compounds. Interestingly, the correlations found between the physico-chemical parameters of the selected compounds and those of commercial molecules that target specific organelles indicate that our rationale might be helpful to drive compound design toward high activities and acceptable pharmacokinetic properties for all compound families.

MycPermCheck: the Mycobacterium tuberculosis permeability prediction tool for small molecules.

MOTIVATION: With >8 million new cases in 2010, particularly documented in developing countries, tuberculosis (TB) is still a highly present pandemic and often terminal. This is also due to the emergence of antibiotic-resistant strains (MDR-TB and XDR-TB) of the primary causative TB agent Mycobacterium tuberculosis (MTB). Efforts to develop new effective drugs against MTB are restrained by the unique and largely impermeable composition of the mycobacterial cell wall. RESULTS: Based on a database of antimycobacterial substances (CDD TB), 3815 compounds were classified as active and thus permeable. A data mining approach was conducted to gather the physico-chemical similarities of these substances and delimit them from a generic dataset of drug-like molecules. On the basis of the differences in these datasets, a regression model was generated and implemented into the online tool MycPermCheck to predict the permeability probability of small organic compounds. DISCUSSION: Given the current lack of precise molecular criteria determining mycobacterial permeability, MycPermCheck represents an unprecedented prediction tool intended to support antimycobacterial drug discovery. It follows a novel knowledge-driven approach to estimate the permeability probability of small organic compounds. As such, MycPermCheck can be used intuitively as an additional selection criterion for potential new inhibitors against MTB. Based on the validation results, its performance is expected to be of high practical value for virtual screening purposes. AVAILABILITY: The online tool is freely accessible under the URL http://www.mycpermcheck.aksotriffer.pharmazie.uni-wuerzburg.de

Protocol for rational design of covalently interacting inhibitors.

The inhibition potencies of covalent inhibitors mainly result from the formation of a covalent bond to the enzyme during the inhibition mechanism. This class of inhibitors has essentially been ignored in previous target-directed drug discovery projects because of concerns about possible side effects. However, their advantages, such as higher binding energies and longer drug-target residence times moved them into the focus of recent investigations. While the rational design of non-covalent inhibitors became standard the corresponding design of covalent inhibitors is still in its early stages. Potent covalent inhibitors can be retrieved from large compound libraries by covalent docking approaches but protocols are missing that can reliably predict the influence of variations in the substitution pattern on the affinity and/or reactivity of a given covalent inhibitor. Hence, the wanted property profile can only be obtained from trial-and-error proceedings. This paper presents an appropriate protocol which is able to predict improved covalent inhibitors. It uses hybrid approaches, which mix quantum mechanical (QM) and molecular mechanical (MM) methods to predict variations in the reactivity of the inhibitor. They are also used to compute the required information about the non-covalent enzyme-inhibitor complex. Docking tools are employed to improve the inhibitor with respect to the non-covalent interactions formed in the binding site.

Amine substitution of quinazolinones leads to selective nanomolar AChE inhibitors with 'inverted' binding mode.

Selective and nanomolar acetylcholinesterase inhibitors were obtained by connecting tri- and tetracyclic quinazolinones-previously described as moderately active and unselective cholinesterase (ChE) inhibitors-via a hydroxyl group in para position to an anilinic nitrogen with different amines linked via a three carbon atom spacer. These tri- and tetracyclic quinazolinones containing different alicyclic ring sizes and connected to tertiary amines were docked to a high-resolution hAChE crystal structure to investigate the preferred binding mode in relation to results obtained by experimental structure-activity relationships. While the 'classical orientation' locating the heterocycle in the active site was rarely found, an alternative binding mode with the basic aliphatic amine in the active center ('inverted' orientation) was obtained for most compounds. Analyses of extended SARs based on this inverted binding mode are able to explain the compounds' binding affinities at AChE.

Rational optimization of drug-target residence time: insights from inhibitor binding to the Staphylococcus aureus FabI enzyme-product complex.

Drug-target kinetics has recently emerged as an especially important facet of the drug discovery process. In particular, prolonged drug-target residence times may confer enhanced efficacy and selectivity in the open in vivo system. However, the lack of accurate kinetic and structural data for a series of congeneric compounds hinders the rational design of inhibitors with decreased off-rates. Therefore, we chose the Staphylococcus aureus enoyl-ACP reductase (saFabI)--an important target for the development of new anti-staphylococcal drugs--as a model system to rationalize and optimize the drug-target residence time on a structural basis. Using our new, efficient, and widely applicable mechanistically informed kinetic approach, we obtained a full characterization of saFabI inhibition by a series of 20 diphenyl ethers complemented by a collection of 9 saFabI-inhibitor crystal structures. We identified a strong correlation between the affinities of the investigated saFabI diphenyl ether inhibitors and their corresponding residence times, which can be rationalized on a structural basis. Because of its favorable interactions with the enzyme, the residence time of our most potent compound exceeds 10 h. In addition, we found that affinity and residence time in this system can be significantly enhanced by modifications predictable by a careful consideration of catalysis. Our study provides a blueprint for investigating and prolonging drug-target kinetics and may aid in the rational design of long-residence-time inhibitors targeting the essential saFabI enzyme.

SFCscore(RF): a random forest-based scoring function for improved affinity prediction of protein-ligand complexes.

A major shortcoming of empirical scoring functions for protein-ligand complexes is the low degree of correlation between predicted and experimental binding affinities, as frequently observed not only for large and diverse data sets but also for SAR series of individual targets. Improvements can be envisaged by developing new descriptors, employing larger training sets of higher quality, and resorting to more sophisticated regression methods. Herein, we describe the use of SFCscore descriptors to develop an improved scoring function by means of a PDBbind training set of 1005 complexes in combination with random forest for regression. This provided SFCscore(RF) as a new scoring function with significantly improved performance on the PDBbind and CSAR-NRC HiQ benchmarks in comparison to previously developed SFCscore functions. A leave-cluster-out cross-validation and performance in the CSAR 2012 scoring exercise point out remaining limitations but also directions for further improvements of SFCscore(RF) and empirical scoring functions in general.

Structure-based search for new inhibitors of cholinesterases.

Cholinesterases are important biological targets responsible for regulation of cholinergic transmission, and their inhibitors are used for the treatment of Alzheimer's disease. To design new cholinesterase inhibitors, of different structure-based design strategies was followed, including the modification of compounds from a previously developed library and a fragment-based design approach. This led to the selection of heterodimeric structures as potential inhibitors. Synthesis and biological evaluation of selected candidates confirmed that the designed compounds were acetylcholinesterase inhibitors with IC50 values in the mid-nanomolar to low micromolar range, and some of them were also butyrylcholinesterase inhibitors.

Probing the dynamic nature of water molecules and their influences on ligand binding in a model binding site.

The model binding site of the cytochrome c peroxidase (CCP) W191G mutant is used to investigate the structural and dynamic properties of the water network at the buried cavity using computational methods supported by crystallographic analysis. In particular, the differences of the hydration pattern between the uncomplexed state and various complexed forms are analyzed as well as the differences between five complexes of CCP W191G with structurally closely related ligands. The ability of docking programs to correctly handle the water molecules in these systems is studied in detail. It is found that fully automated prediction of water replacement or retention upon docking works well if some additional preselection is carried out but not necessarily if the entire water network in the cavity is used as input. On the other hand, molecular interaction fields for water calculated from static crystal structures and hydration density maps obtained from molecular dynamics simulations agree very well with crystallographically observed water positions. For one complex, the docking and MD results sensitively depend on the quality of the starting structure, and agreement is obtained only after redetermination of the crystal structure and refinement at higher resolution.

Molecular dynamics of Mycobacterium tuberculosis KasA: implications for inhibitor and substrate binding and consequences for drug design.

Inhibition of the production of fatty acids as essential components of the mycobacterial cell wall has been an established way of fighting tuberculosis for decades. However, increasing resistances and an outdated medical treatment call for the validation of new targets involved in this crucial pathway. In this regard, the ß-ketoacyl ACP synthase KasA is a promising enzyme. In this study, three molecular dynamics simulations based on the wildtype crystal structures of inhibitor bound and unbound KasA were performed in order to investigate the flexibility and conformational space of this target. We present an exhaustive analysis of the binding-site flexibility and representative pocket conformations that may serve as new starting points for structure-based drug design. We also revealed a mechanism which may account for the comparatively low binding affinity of thiolactomycin. Furthermore, we examined the behavior of water molecules within the binding pocket and provide recommendations how to handle them in the drug design process. Finally, we analyzed the dynamics of a channel that accommodates the long-chain fatty acid substrates and, thereby, propose a mechanism of substrate access to this channel and how products are most likely released.

Molecular dynamics simulations of the HIV-1 integrase dimerization interface: guidelines for the design of a novel class of integrase inhibitors.

HIV-1 integrase (IN) is a validated target of anti-AIDS research. The classical approach of designing active-site directed ligands has largely been exploited. A promising alternative strategy to inactivate the enzyme is to prevent the formation of IN dimers. The rational design of dimerization inhibitors, however, is hampered by the lack of relevant structural data about the targeted monomeric form. Therefore, we performed molecular dynamics simulations and subsequent analyses to gain insight into the structural features of the IN catalytic-core-domain dimerization interface. As a result, the formation of a groove and a cavity along the dimerization interface of the IN monomer could be revealed. Both were shown to be suited for accommodating an inhibitory peptide. The results form a valuable basis for the design of ligands targeting the dimerization interface and, thus, of a whole new class of HIV-1 integrase inhibitors.

SFCscore: scoring functions for affinity prediction of protein-ligand complexes.

Empirical scoring functions to calculate binding affinities of protein-ligand complexes have been calibrated based on experimental structure and affinity data collected from public and industrial sources. Public data were taken from the AffinDB database, whereas access to industrial data was gained through the Scoring Function Consortium (SFC), a collaborative effort with various pharmaceutical companies and the Cambridge Crystallographic Data Center. More than 850 complexes were obtained by the data collection procedure and subsequently used to setup different training sets for the parameterization of new scoring functions. Over 60 different descriptors were evaluated for all complexes, including terms accounting for interactions with and among aromatic ring systems as well as many surface-dependent terms. After exploratory correlation and regression analyses, stepwise variable selection procedures and systematic searches, the most suitable descriptors were chosen as variables to calibrate regression functions by means of multiple linear regression or partial least squares analysis. Eight different functions are presented herein. Cross-validated r(2) (Q(2)) values of up to 0.72 and standard errors (s(PRESS)) generally below 1.15 pK(i) units suggest highly predictive functions. Extensive unbiased validation was carried out by testing the functions on large data sets from the PDBbind database as used by Wang et al. (J Chem Inf Comput Sci 2004;44:2114-2125) in a comparative analysis of other scoring functions. Superior performance of the SFCscore functions is observed in many cases, but the results also illustrate the need for further improvements.

Protonation changes upon ligand binding to trypsin and thrombin: structural interpretation based on pK(a) calculations and ITC experiments.

The protonation states of a protein and a ligand can be altered upon complex formation. Such changes can be detected experimentally by isothermal titration calorimetry (ITC). For a series of ligands binding to the serine proteases trypsin and thrombin, we previously performed an extensive ITC and crystallographic study and were able to identify protonation changes for four complexes. However, since ITC measures only the overall proton exchange, it does not provide structural insights into the functional groups involved in the proton transfer. Using Poisson-Boltzmann calculations based on our recently developed PEOE_PB charges, we compute pK(a) values for all complexes of our former study in order to reveal the residues with altered protonation states. The results indicate that His57, a member of the catalytic triad, is responsible for the most relevant pK(a) shifts leading to the experimentally detected protonation changes. This finding is in contrast to our previous assumption that the observed protonation changes occur at the carboxylic group of the ligands. The newly detected proton acceptor is used for a revised factorization of the ITC data, which is necessary whenever the protonation inventory changes upon complexation. The pK(a) values of complexes showing no protonation change in the ITC experiment are reliably predicted in most cases, whereas predictions of strongly coupled systems remain problematic.

Tracing changes in protonation: a prerequisite to factorize thermodynamic data of inhibitor binding to aldose reductase.

To prevent diabetic complications derived from enhanced glucose flux via the polyol pathway the development of aldose reductase inhibitors (ARIs) has been established as a promising therapeutic concept. Here, we study the binding process of inhibitors to aldose reductase (ALR2) with respect to changes of the protonation inventory upon complex formation. Knowledge of such processes is a prerequisite to factorize the binding free energy into enthalpic and entropic contributions on an absolute scale. Our isothermal titration calorimetry (ITC) measurements suggest a proton uptake upon complex formation with carboxylate-type inhibitors. As the protonation event will contribute strongly to the enthalpic signal recorded during ITC experiments, knowledge about the proton-accepting and releasing functional groups of the system is of utmost importance. However, this is intricate to retrieve, if, as in the present case, both, binding site and ligand possess several titratable groups. Here, we present pKa calculations complemented by mutagenesis and thermodynamic measurements suggesting a tyrosine residue located in the catalytic site (Tyr48) as a likely candidate to act as proton acceptor upon inhibitor binding, as it occurs deprotonated to a remarkable extent if only the cofactor NADP+ is bound. We furthermore provide evidence that the protonation state and binding thermodynamics depend strongly on the oxidation state of the cofactor;s nicotinamide moiety. Binding thermodynamics of IDD 388, IDD 393, tolrestat, sorbinil, and fidarestat are discussed in the context of substituent effects.

AffinDB: a freely accessible database of affinities for protein-ligand complexes from the PDB.

AffinDB is a database of affinity data for structurally resolved protein-ligand complexes from the Protein Data Bank (PDB). It is freely accessible at http://www.agklebe.de/affinity. Affinity data are collected from the scientific literature, both from primary sources describing the original experimental work of affinity determination and from secondary references which report affinity values determined by others. AffinDB currently contains over 730 affinity entries covering more than 450 different protein-ligand complexes. Besides the affinity value, PDB summary information and additional data are provided, including the experimental conditions of the affinity measurement (if available in the corresponding reference); 2D drawing, SMILES code and molecular weight of the ligand; links to other databases, and bibliographic information. AffinDB can be queried by PDB code or by any combination of affinity range, temperature and pH value of the measurement, ligand molecular weight, and publication data (author, journal and year). Search results can be saved as tabular reports in text files. The database is supposed to be a valuable resource for researchers interested in biomolecular recognition and the development of tools for correlating structural data with affinities, as needed, for example, in structure-based drug design.

Expect the unexpected or caveat for drug designers: multiple structure determinations using aldose reductase crystals treated under varying soaking and co-crystallisation conditions.

In structure-based drug design, accurate crystal structure determination of protein-ligand complexes is of utmost importance in order to elucidate the binding characteristics of a putative lead to a given target. It is the starting point for further design hypotheses to predict novel leads with improved properties. Often, crystal structure determination is regarded as ultimate proof for ligand binding providing detailed insight into the specific binding mode of the ligand to the protein. This widely accepted practise relies on the assumption that the crystal structure of a given protein-ligand complex is unique and independent of the protocol applied to produce the crystals. We present two examples indicating that this assumption is not generally given, even though the composition of the mother liquid for crystallisation was kept unchanged: Multiple crystal structure determinations of aldose reductase complexes obtained under varying crystallisation protocols concerning soaking and crystallisation exposure times were performed resulting in a total of 17 complete data sets and ten refined crystal structures, eight in complex with zopolrestat and two complexed with tolrestat. In the first example, a flip of a peptide bond is observed, obviously depending on the crystallisation protocol with respect to soaking and co-crystallisation conditions. This peptide flip is accompanied by a rupture of an H-bond formed to the bound ligand zopolrestat. The indicated enhanced local mobility of the complex is in agreement with the results of molecular dynamics simulations. As a second example, the aldose reductase-tolrestat complex is studied. Unexpectedly, two structures could be obtained: one with one, and a second with four inhibitor molecules bound to the protein. They are located in and near the binding pocket facilitated by crystal packing effects. Accommodation of the four ligand molecules is accompanied by pronounced shifts concerning two helices interacting with the additional ligands.