A virtual library of small molecules mimicking dipeptides.

Virtual combinatorial libraries are prevalent in drug discovery due to improvements in the prediction of synthetic reactions that can be performed. This has gone hand in hand with the development of virtual screening capabilities to effectively screen the large chemical spaces spanned by exhaustive enumeration of reaction products. In this study, we generated a small-molecule dipeptide mimic library to target proteins binding small peptides. The library was created based on the general idea of peptide synthesis, that is, amino acid mimics were reacted in silico to form the dipeptide mimics, yielding 2,036,819 unique compounds. After docking calculations, two compounds from the library were synthesized and tested against WD repeat-containing protein 5 (WDR5) and histamine receptors H1-H4 to evaluate whether these molecules are viable in assays. The compounds showed the highest potency at the histamine H3 receptor, with Ki values in the two-digit micromolar range.

Structural and Biochemical Investigation of the Heterodimeric Murine tRNA-Guanine Transglycosylase.

In tRNAAsp, tRNAAsn, tRNATyr, and tRNAHis of most bacteria and eukaryotes, the anticodon wobble position may be occupied by the modified nucleoside queuosine, which affects the speed and the accuracy of translation. Since eukaryotes are not able to synthesize queuosine de novo, they have to salvage queuine (the queuosine base) as a micronutrient from food and/or the gut microbiome. The heterodimeric Zn2+ containing enzyme tRNA-guanine transglycosylase (TGT) catalyzes the insertion of queuine into the above-named tRNAs in exchange for the genetically encoded guanine. This enzyme has attracted medical interest since it was shown to be potentially useful for the treatment of multiple sclerosis. In addition, TGT inactivation via gene knockout leads to the suppressed cell proliferation and migration of certain breast cancer cells, which may render this enzyme a potential target for the design of compounds supporting breast cancer therapy. As a prerequisite to fully exploit the medical potential of eukaryotic TGT, we have determined and analyzed a number of crystal structures of the functional murine TGT with and without bound queuine. In addition, we have investigated the importance of two residues of its non-catalytic subunit on dimer stability and determined the Michaelis-Menten parameters of murine TGT with respect to tRNA and several natural and artificial nucleobase substrates. Ultimately, on the basis of available TGT crystal structures, we provide an entirely conclusive reaction mechanism for this enzyme, which in detail explains why the TGT-catalyzed insertion of some nucleobases into tRNA occurs reversibly while that of others is irreversible.

Frag4Lead: growing crystallographic fragment hits by catalog using fragment-guided template docking.

In recent years, crystallographic fragment screening has matured into an almost routine experiment at several modern synchrotron sites. The hits of the screening experiment, i.e. small molecules or fragments binding to the target protein, are revealed along with their 3D structural information. Therefore, they can serve as useful starting points for further structure-based hit-to-lead development. However, the progression of fragment hits to tool compounds or even leads is often hampered by a lack of chemical feasibility. As an attractive alternative, compound analogs that embed the fragment hit structurally may be obtained from commercial catalogs. Here, a workflow is reported based on filtering and assessing such potential follow-up compounds by template docking. This means that the crystallographic binding pose was integrated into the docking calculations as a central starting parameter. Subsequently, the candidates are scored on their interactions within the binding pocket. In an initial proof-of-concept study using five starting fragments known to bind to the aspartic protease endothiapepsin, 28 follow-up compounds were selected using the designed workflow and their binding was assessed by crystallography. Ten of these compounds bound to the active site and five of them showed significantly increased affinity in isothermal titration calorimetry of up to single-digit micromolar affinity. Taken together, this strategy is capable of efficiently evolving the initial fragment hits without major synthesis efforts and with full control by X-ray crystallography.

Surprising Non-Additivity of Methyl Groups in Drug-Kinase Interaction.

Drug optimization is guided by biophysical methods with increasing popularity. In the context of lead structure modifications, the introduction of methyl groups is a simple but potentially powerful approach. Hence, it is crucial to systematically investigate the influence of ligand methylation on biophysical characteristics such as thermodynamics. Here, we investigate the influence of ligand methylation in different positions and combinations on the drug-kinase interaction. Binding modes and complex structures were analyzed using protein crystallography. Thermodynamic signatures were measured via isothermal titration calorimetry (ITC). An extensive computational analysis supported the understanding of the underlying mechanisms. We found that not only position but also stereochemistry of the methyl group has an influence on binding potency as well as the thermodynamic signature of ligand binding to the protein. Strikingly, the combination of single methyl groups does not lead to additive effects. In our case, the merger of two methyl groups in one ligand leads to an entirely new alternative ligand binding mode in the protein ligand complex. Moreover, the combination of the two methyl groups also resulted in a nonadditive thermodynamic profile of ligand binding. Molecular dynamics (MD) simulations revealed distinguished characteristic motions of the ligands in solution explaining the pronounced thermodynamic changes. The unexpected drastic change in protein ligand interaction highlights the importance of crystallographic control even for minor modifications such as the introduction of a methyl group. For an in-depth understanding of ligand binding behavior, MD simulations have shown to be a powerful tool.

Structural insights into the stimulation of S. pombe Dnmt2 catalytic efficiency by the tRNA nucleoside queuosine.

Dnmt2 methylates cytosine at position 38 of tRNAAsp in a variety of eukaryotic organisms. A correlation between the presence of the hypermodified nucleoside queuosine (Q) at position 34 of tRNAAsp and the Dnmt2 dependent C38 methylation was recently found in vivo for S. pombe and D. discoideum. We demonstrate a direct effect of the Q-modification on the methyltransferase catalytic efficiency in vitro, as Vmax/K0.5 of purified S. pombe Dnmt2 shows an increase for in vitro transcribed tRNAAsp containing Q34 to 6.27 ∗ 10-3 s-1 µM-1 compared to 1.51 ∗ 10-3 s-1 µM-1 for the unmodified substrate. Q34tRNAAsp exhibits an only slightly increased affinity for Dnmt2 in comparison to unmodified G34tRNA. In order to get insight into the structural basis for the Q-dependency, the crystal structure of S. pombe Dnmt2 was determined at 1.7 Å resolution. It closely resembles the known structures of human and E. histolytica Dnmt2, and contains the entire active site loop. The interaction with tRNA was analyzed by means of mass-spectrometry using UV cross-linked Dnmt2-tRNA complex. These cross-link data and computational docking of Dnmt2 and tRNAAsp reveal Q34 positioned adjacent to the S-adenosylmethionine occupying the active site, suggesting that the observed increase of Dnmt2 catalytic efficiency by queuine originates from optimal positioning of the substrate molecules and residues relevant for methyl transfer.

Paradoxically, Most Flexible Ligand Binds Most Entropy-Favored: Intriguing Impact of Ligand Flexibility and Solvation on Drug-Kinase Binding.

Biophysical parameters can accelerate drug development; e.g., rigid ligands may reduce entropic penalty and improve binding affinity. We studied systematically the impact of ligand rigidification on thermodynamics using a series of fasudil derivatives inhibiting protein kinase A by crystallography, isothermal titration calorimetry, nuclear magnetic resonance, and molecular dynamics simulations. The ligands varied in their internal degrees of freedom but conserve the number of heteroatoms. Counterintuitively, the most flexible ligand displays the entropically most favored binding. As experiment shows, this cannot be explained by higher residual flexibility of ligand, protein, or formed complex nor by a deviating or increased release of water molecules upon complex formation. NMR and crystal structures show no differences in flexibility and water release, although strong ligand-induced adaptations are observed. Instead, the flexible ligand entraps more efficiently water molecules in solution prior to protein binding, and by release of these waters, the favored entropic binding is observed.

Charges Shift Protonation: Neutron Diffraction Reveals that Aniline and 2-Aminopyridine Become Protonated Upon Binding to Trypsin.

Hydrogen atoms play a key role in protein-ligand recognition. They determine the quality of established H-bonding networks and define the protonation of bound ligands. Structural visualization of H atoms by X-ray crystallography is rarely possible. We used neutron diffraction to determine the positions of the hydrogen atoms in the ligands aniline and 2-aminopyridine bound to the archetypical serine protease trypsin. The resulting structures show the best resolution so far achieved for proteins larger than 100 residues and allow an accurate description of the protonation states and interactions with nearby water molecules. Despite its low pKa of 4.6 and a large distance of 3.6 Što the charged Asp189 at the bottom of the S1 pocket, the amino group of aniline becomes protonated, whereas in 2-aminopyridine, the pyridine nitrogen picks up the proton although its amino group is 1.6 Šcloser to Asp189. Therefore, apart from charge-charge distances, tautomer stability is decisive for the resulting binding poses, an aspect that is pivotal for predicting correct binding.

Kinetic and Structural Insights into the Mechanism of Binding of Sulfonamides to Human Carbonic Anhydrase by Computational and Experimental Studies.

The binding of sulfonamides to human carbonic anhydrase II (hCAII) is a complex and long-debated example of protein-ligand recognition and interaction. In this study, we investigate the para-substituted n-alkyl and hydroxyethylene-benzenesulfonamides, providing a complete reconstruction of their binding pathway to hCAII by means of large-scale molecular dynamics simulations, density functional calculations, surface plasmon resonance (SPR) measurements, and X-ray crystallography experiments. Our analysis shows that the protein-ligand association rate (kon) dramatically increases with the ligand's hydrophobicity, pointing to the existence of a prebinding stage largely stabilized by a favorable packing of the ligand's apolar moieties with the hCAII "hydrophobic wall". The characterization of the binding pathway allows an unprecedented understanding of the structure-kinetic relationship in hCAII/benzenesulfonamide complexes, depicting a paradigmatic scenario for the multistep binding process in protein-ligand systems.

Frontiers in Medicinal Chemistry 2015: Meet the Experts of MedChem near the Cradle of Medicinal and Pharmaceutical Chemistry.

Pioneering inspiration: Right next to the former laboratories of Johannes Hartmann, the first so-called "Professor of Chymiatrie", the 2015 Frontiers in Medicinal Chemistry meeting was held last March at Philipps University in Marburg, Germany. Herein we give readers an idea of what it was like to attend the conference, which was organized jointly by the DPhG, GDCh, and SCS. Along with the lectures, we also describe the poster sessions, social program, and awards.

Investigation of specificity determinants in bacterial tRNA-guanine transglycosylase reveals queuine, the substrate of its eucaryotic counterpart, as inhibitor.

Bacterial tRNA-guanine transglycosylase (Tgt) catalyses the exchange of the genetically encoded guanine at the wobble position of tRNAs(His,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.

Concise and efficient syntheses of preQ1 base, Q base, and (ent)-Q base.

To thoroughly study the functional role of prokaryotic t-RNA-guanine-transglycosylases which are essential in the pathogenesis of shigellosis, novel efficient, high-yielding synthetic approaches for preQ(1) base, Q base, as well as for (ent)-Q base mainly employing cheap and readily available starting materials have been developed. Q base as well as (ent)-Q base are accessible starting from preQ(1) base via nucleophilic substitution reactions with appropriately decorated halocyclopentenyl synthons, prior to that prepared from naturally occurring carbohydrates.

Fragment-based lead discovery: screening and optimizing fragments for thermolysin inhibition.

Fragment-based drug discovery has gained a foothold in today's lead identification processes. We present the application of in silico fragment-based screening for the discovery of novel lead compounds for the metalloendoproteinase thermolysin. We have chosen thermolysin to validate our screening approach as it is a well-studied enzyme and serves as a model system for other proteases. A protein-targeted virtual library was designed and screening was carried out using the program AutoDock. Two fragment hits could be identified. For one of them, the crystal structure in complex with thermolysin is presented. This compound was selected for structure-based optimization of binding affinity and improvement of ligand efficiency, while concomitantly keeping the fragment-like properties of the initial hit. Redesigning the zinc coordination group revealed a novel class of fragments possessing K(i) values as low as 128 microM, thus they provide a good starting point for further hit evolution in a tailored lead design.

Proteases of Plasmodium falciparum as potential drug targets and inhibitors thereof.

Malaria, caused by protozoa of the genus Plasmodium, remains one of the most dreadful infectious diseases worldwide killing more than 1 million people per year. The emergence of multidrug-resistant parasites highly demands a steadfast and continuous search not only for new targets but also for new anti-infectives addressing the known ones. As proteases in general have been proven to be excellent drug targets and the development of inhibitors has frequently resulted in approved drugs, this review will only focus on the proteases of Plasmodium falciparum as drug targets. The completion of the sequencing of the Plasmodium falciparum genome in 2002 lead to the discovery of nearly 100 putative proteases encoded therein. Within this review, only those proteases and inhibitors thereof will be discussed in more detail, in which their biological function has been determined undoubtedly or in those cases, in which the development of specific inhibitors has significantly contributed to the understanding of the underlying biological role of the respective protease thus validating the role as promising drug target.

Virtual screening for submicromolar leads of tRNA-guanine transglycosylase based on a new unexpected binding mode detected by crystal structure analysis.

Eubacterial tRNA-guanine transglycosylase (TGT) is involved in the hypermodification of cognate tRNAs, leading to the exchange of G34 by preQ1 at the wobble position in the anticodon loop. Mutation of the tgt gene in Shigella flexneri results in a significant loss of pathogenicity of the bacterium due to inefficient translation of a virulence protein mRNA. Herein, we describe the discovery of a ligand with an unexpected binding mode. On the basis of this binding mode, three slightly deviating pharmacophore hypotheses have been derived. Virtual screening based on this composite pharmacophore model retrieved a set of potential TGT inhibitors belonging to several compound classes. All nine tested inhibitors being representatives of these classes showed activity in the micromolar range, two of them even in the submicromolar range.