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1.  Structure-Based Optimization of Inhibitors of the Aspartic Protease Endothiapepsin 
Aspartic proteases are a class of enzymes that play a causative role in numerous diseases such as malaria (plasmepsins), Alzheimer’s disease (β-secretase), fungal infections (secreted aspartic proteases), and hypertension (renin). We have chosen endothiapepsin as a model enzyme of this class of enzymes, for the design, preparation and biochemical evaluation of a new series of inhibitors of endothiapepsin. Here, we have optimized a hit, identified by de novo structure-based drug design (SBDD) and DCC, by using structure-based design approaches focusing on the optimization of an amide–π interaction. Biochemical results are in agreement with SBDD. These results will provide useful insights for future structure-based optimization of inhibitors for the real drug targets as well as insights into molecular recognition.
PMCID: PMC4581293  PMID: 26287174
inhibitors; aspartic protease endothiapepsin; structure-based drug design; molecular recognition
4.  Novel Type II Fatty Acid Biosynthesis (FAS II) Inhibitors as Multistage Antimalarial Agents 
ChemMedChem  2013;8(3):442-461.
Malaria is a potentially fatal disease caused by Plasmodium parasites and poses a major medical risk in large parts of the world. The development of new, affordable antimalarial drugs is of vital importance as there are increasing reports of resistance to the currently available therapeutics. In addition, most of the current drugs used for chemoprophylaxis merely act on parasites already replicating in the blood. At this point, a patient might already be suffering from the symptoms associated with the disease and could additionally be infectious to an Anopheles mosquito. These insects act as a vector, subsequently spreading the disease to other humans. In order to cure not only malaria but prevent transmission as well, a drug must target both the blood- and pre-erythrocytic liver stages of the parasite. P. falciparum (Pf) enoyl acyl carrier protein (ACP) reductase (ENR) is a key enzyme of plasmodial type II fatty acid biosynthesis (FAS II). It has been shown to be essential for liver-stage development of Plasmodium berghei and is therefore qualified as a target for true causal chemoprophylaxis. Using virtual screening based on two crystal structures of PfENR, we identified a structurally novel class of FAS inhibitors. Subsequent chemical optimization yielded two compounds that are effective against multiple stages of the malaria parasite. These two most promising derivatives were found to inhibit blood-stage parasite growth with IC50 values of 1.7 and 3.0 µm and lead to a more prominent developmental attenuation of liver-stage parasites than the gold-standard drug, primaquine.
PMCID: PMC3633473  PMID: 23341167
antimalarial agents; fatty acid biosynthesis; molecular modeling; multistage inhibitors; Plasmodium falciparum; virtual screening
5.  Targeting the Blind Spot of Polycationic Nanocarrier-Based siRNA Delivery 
ACS nano  2012;6(11):10.1021/nn301966r.
Polycationic nanocarriers attract increasing attention to the field of siRNA delivery. We investigated the self-assembly of siRNA vs pDNA with polycations, which are broadly used for nonviral gene and siRNA delivery. Although polyethyleneimine (PEI) was routinely adopted as siRNA carrier based on its efficacy in delivering pDNA, it has not been investigated yet why PEI efficiently delivers pDNA to cells but is controversially discussed in terms of efficacy for siRNA delivery. We are the first to investigate the self-assembly of PEI/siRNA vs PEI/pDNA and the steps of complexation and aggregation through different levels of hierarchy on the atomic and molecular scale with the novel synergistic use of molecular modeling, molecular dynamics simulation, isothermal titration calorimetry, and other characterization techniques. We are also the fist to elucidate atomic interactions, size, shape, stoichiometry, and association dynamics for polyplexes containing siRNA vs pDNA. Our investigation highlights differences in the hierarchical mechanism of formation of related polycation–siRNA and polycation–pDNA complexes. The results of fluorescence quenching assays indicated a biphasic behavior of siRNA binding with polycations where molecular reorganization of the siRNA within the polycations occurred at lower N/P ratios (nitrogen/phosphorus). Our results, for the first time, emphasize a biphasic behavior in siRNA complexation and the importance of low N/P ratios, which allow for excellent siRNA delivery efficiency. Our investigation highlights the formulation of siRNA complexes from a thermodynamic point of view and opens new perspectives to advance the rational design of new siRNA delivery systems.
PMCID: PMC3882193  PMID: 23036046
siRNA delivery; DNA delivery; polyethyleneimine; molecular modeling; isothermal titration calorimetry; RT-PCR; supramolecular complexation
6.  Investigation of Specificity Determinants in Bacterial tRNA-Guanine Transglycosylase Reveals Queuine, the Substrate of Its Eucaryotic Counterpart, as Inhibitor 
PLoS ONE  2013;8(5):e64240.
Bacterial tRNA-guanine transglycosylase (Tgt) catalyses the exchange of the genetically encoded guanine at the wobble position of tRNAsHis,Tyr,Asp,Asn by the premodified base preQ1, which is further converted to queuine at the tRNA level. As eucaryotes are not able to synthesise queuine de novo but acquire it through their diet, eucaryotic Tgt directly inserts the hypermodified base into the wobble position of the tRNAs mentioned above. Bacterial Tgt is required for the efficient pathogenicity of Shigella sp, the causative agent of bacillary dysentery and, hence, it constitutes a putative target for the rational design of anti-Shigellosis compounds. Since mammalian Tgt is known to be indirectly essential to the conversion of phenylalanine to tyrosine, it is necessary to create substances which only inhibit bacterial but not eucaryotic Tgt. Therefore, it seems of utmost importance to study selectivity-determining features within both types of proteins. Homology models of Caenorhabditis elegans Tgt and human Tgt suggest that the replacement of Cys158 and Val233 in bacterial Tgt (Zymomonas mobilis Tgt numbering) by valine and accordingly glycine in eucaryotic Tgt largely accounts for the different substrate specificities. In the present study we have created mutated variants of Z. mobilis Tgt in order to investigate the impact of a Cys158Val and a Val233Gly exchange on catalytic activity and substrate specificity. Using enzyme kinetics and X-ray crystallography, we gained evidence that the Cys158Val mutation reduces the affinity to preQ1 while leaving the affinity to guanine unaffected. The Val233Gly exchange leads to an enlarged substrate binding pocket, that is necessary to accommodate queuine in a conformation compatible with the intermediately covalently bound tRNA molecule. Contrary to our expectations, we found that a priori queuine is recognised by the binding pocket of bacterial Tgt without, however, being used as a substrate.
PMCID: PMC3660597  PMID: 23704982
8.  Bidentate Zinc Chelators for α-Carbonic Anhydrases that Produce a Trigonal Bipyramidal Coordination Geometry 
ChemMedChem  2010;5(9):1609-1615.
A series of new zinc binding groups (ZBGs) has been evaluated kinetically on 13 carbonic anhydrase (CA) isoforms. The fragments show affinity for all isoforms with IC50 values in the range of 2–11 µm. The crystal structure of hCA II in complex with one such fragment reveals a bidentate binding mode with a trigonal-bipyramidal coordination geometry at the Zn2+ center. The fragment also interacts with Thr199 and Thr200 through hydrogen bonding and participates in a water network. Further development of this ZBG should increase the binding affinity leading to a structurally distinct and promising class of CA inhibitors.
PMCID: PMC3019337  PMID: 20629007
carbonic anhydrase; crystal structures; drug discovery; inhibitors; metalloenzymes; zinc binding group
9.  On the validity of popular assumptions in computational drug design 
Journal of Cheminformatics  2011;3(Suppl 1):O18.
PMCID: PMC3083548
10.  Molecular Docking Screens Using Comparative Models of Proteins 
Two orders of magnitude more protein sequences can be modeled by comparative modeling than have been determined by X-ray crystallography and NMR spectroscopy. Investigators have nevertheless been cautious about using comparative models for ligand discovery because of concerns about model errors. We suggest how to exploit comparative models for molecular screens, based on docking against a wide range of crystallographic structures and comparative models with known ligands. To account for the variation in the ligand-binding pocket as it binds different ligands, we calculate “consensus” enrichment by ranking each library compound by its best docking score against all available comparative models and/or modeling templates. For the majority of the targets, the consensus enrichment for multiple models was better or comparable to that of the holo and apo X-ray structures. Even for single models, the models are significantly more enriching than the template structure if the template is paralogous and shares more than 25% sequence identity with the target.
PMCID: PMC2790034  PMID: 19845314
comparative modeling; docking screens; consensus enrichment
11.  A Structure-Based Approach to Ligand Discovery for 2C-Methyl-d-erythritol-2,4-cyclodiphosphate Synthase: A Target for Antimicrobial Therapy† 
Journal of Medicinal Chemistry  2009;52(8):2531-2542.
The nonmevalonate route to isoprenoid biosynthesis is essential in Gram-negative bacteria and apicomplexan parasites. The enzymes of this pathway are absent from mammals, contributing to their appeal as chemotherapeutic targets. One enzyme, 2C-methyl-d-erythritol-2,4-cyclodiphosphate synthase (IspF), has been validated as a target by genetic approaches in bacteria. Virtual screening against Escherichia coli IspF (EcIspF) was performed by combining a hierarchical filtering methodology with molecular docking. Docked compounds were inspected and 10 selected for experimental validation. A surface plasmon resonance assay was developed and two weak ligands identified. Crystal structures of EcIspF complexes were determined to support rational ligand development. Cytosine analogues and Zn2+-binding moieties were characterized. One of the putative Zn2+-binding compounds gave the lowest measured KD to date (1.92 ± 0.18 μM). These data provide a framework for the development of IspF inhibitors to generate lead compounds of therapeutic potential against microbial pathogens.
PMCID: PMC2669732  PMID: 19320487
12.  Glutamate versus glutamine exchange swaps substrate selectivity in tRNA-guanine transglycosylase: Insight into the regulation of substrate selectivity by kinetic and crystallographic studies 
Journal of molecular biology  2007;374(3):764-776.
Bacterial tRNA-guanine transglycosylase (Tgt) catalyses the exchange of guanine in the wobble position of particular tRNAs by the modified base preQ1. In vitro, however, the enzyme is also able to insert the immediate biosynthetic precursor, preQ0, into those tRNAs. This substrate promiscuity is based on a peptide switch in the active site, gated by the general acid/base Glu235. The switch alters the properties of the binding pocket to allow either the accommodation of guanine or preQ1. The peptide conformer recognising guanine, however, is also able to bind preQ0. To investigate selectivity regulation, kinetic data for Z. mobilis Tgt were recorded. They show that selectivity in favour of the actual substrate preQ1 over preQ0 is not achieved by a difference in affinity but via a higher turn-over rate. Moreover, a Tgt(Glu235Gln) variant was constructed. The mutation was intended to stabilise the peptide switch in the conformation favouring guanine and preQ0 binding. Kinetic characterisation of the mutated enzyme revealed that the Glu235Gln exchange has, with respect to all substrate bases, no significant influence on kcat. In contrast, KM(preQ1) is drastically increased while KM(preQ0) seems to be decreased. Hence, regarding kcat/KM as an indicator for catalytic efficiency, selectivity of Tgt in favour of preQ1 is abolished or even inverted in favour of preQ0 for Tgt(Glu235Gln). Crystal structures of the mutated enzyme confirm that the mutation strongly favours the binding pocket conformation required for the accommodation of guanine and preQ0. The way this is achieved, however, significantly differs from what was predicted based on crystal structures of wild type Tgt.
PMCID: PMC2100405  PMID: 17949745
peptide flip; induced fit; queuine; queuosine; tRNA modification
14.  PDB2PQR: expanding and upgrading automated preparation of biomolecular structures for molecular simulations 
Nucleic Acids Research  2007;35(Web Server issue):W522-W525.
Real-world observable physical and chemical characteristics are increasingly being calculated from the 3D structures of biomolecules. Methods for calculating pKa values, binding constants of ligands, and changes in protein stability are readily available, but often the limiting step in computational biology is the conversion of PDB structures into formats ready for use with biomolecular simulation software. The continued sophistication and integration of biomolecular simulation methods for systems- and genome-wide studies requires a fast, robust, physically realistic and standardized protocol for preparing macromolecular structures for biophysical algorithms. As described previously, the PDB2PQR web server addresses this need for electrostatic field calculations (Dolinsky et al., Nucleic Acids Research, 32, W665–W667, 2004). Here we report the significantly expanded PDB2PQR that includes the following features: robust standalone command line support, improved pKa estimation via the PROPKA framework, ligand parameterization via PEOE_PB charge methodology, expanded set of force fields and easily incorporated user-defined parameters via XML input files, and improvement of atom addition and optimization code. These features are available through a new web interface (, which offers users a wide range of options for PDB file conversion, modification and parameterization.
PMCID: PMC1933214  PMID: 17488841
15.  AffinDB: a freely accessible database of affinities for protein–ligand complexes from the PDB 
Nucleic Acids Research  2005;34(Database issue):D522-D526.
AffinDB is a database of affinity data for structurally resolved protein–ligand complexes from the Protein Data Bank (PDB). It is freely accessible at . 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.
PMCID: PMC1347402  PMID: 16381925

Results 1-15 (15)