Three different database docking programs (Dock, FlexX, Gold) have been used in combination with seven scoring functions (Chemscore, Dock, FlexX, Fresno, Gold, Pmf, Score) to assess the accuracy of virtual screening methods against two protein targets (thymidine kinase, estrogen receptor) of known three-dimensional structure. For both targets, it was generally possible to discriminate about 7 out of 10 true hits from a random database of 990 ligands. The use of consensus lists common to two or three scoring functions clearly enhances hit rates among the top 5% scorers from 10% (single scoring) to 25−40% (double scoring) and up to 65−70% (triple scoring). However, in all tested cases, no clear relationships could be found between docking and ranking accuracies. Moreover, predicting the absolute binding free energy of true hits was not possible whatever docking accuracy was achieved and scoring function used. As the best docking/consensus scoring combination varies with the selected target and the physicoch...

http://bioinfo-pharma.u-strasbg.fr/labwebsite/publications/paper48.pdf

Protein-based virtual screening of chemical databases. 1. Evaluation of different docking/scoring combinations.

Deoxyribonucleoside kinases phosphorylate deoxyribonucleosides, a crucial reaction in biosynthesis of DNA precursors through the salvage pathway. Their medical interest stems from their activation of a number of anticancer and antiviral drugs such as 2-chloro-2'-deoxyadenosine, azidothymidine and acyclovir. Here we review what is presently known about each of the mammalian kinases as well as some other members of the deoxyribonucleoside kinase family. A description of the biochemical properties of the enzymes is followed by an overview of the structural studies made on this family of enzymes, including the catalytic mechanism as well as the mechanism for feedback inhibition. A presentation of homology models of other proteins in the family is made and, finally, the determinants of substrate and substrate analog specificities are described.

Structure and function of cellular deoxyribonucleoside kinases.

https://courses.washington.edu/pabio568/files/stranska.pdf

Survey of acyclovir-resistant herpes simplex virus in the Netherlands: prevalence and characterization

The presence of water molecules plays an important role in the accuracy of ligand-protein docking predictions. Comprehensive docking simulations have been performed on a large set of ligand-protein complexes whose crystal structures contain water molecules in their binding sites. Only those water molecules found in the immediate vicinity of both the ligand and the protein were considered. We have investigated whether prior optimization of the orientation of water molecules in either the presence or absence of the bound ligand has any effect on the accuracy of docking predictions. We have observed a statistically significant overall increase in accuracy when water molecules are included during docking simulations and have found this to be independent of the method of optimization of the orientation of water molecules. These results confirm the importance of including water molecules whenever possible in a ligand-protein docking simulation. Our findings also reveal that prior optimization of the orientation of water molecules, in the absence of any bound ligand, does not have a detrimental effect on the improved accuracy of ligand-protein docking. This is important, given the use of docking simulations to predict the binding modes of new ligands or drug molecules.

Ligand−Protein Docking with Water Molecules

Multimodality imaging using several reporter genes and imaging technologies has become an increasingly important tool in determining the location(s), magnitude, and time variation of reporter gene expression in small animals. We have reported construction and validation of several triple fusion genes composed of a bioluminescent, a fluorescent, and a positron emission tomography (PET) reporter gene in cell culture and in living subjects. However, the bioluminescent and fluorescent components of fusion reporter proteins encoded by these vectors possess lesser activities when compared with the bioluminescent and fluorescent components of the nonfusions. In this study, we first created a mutant ( mtfl ) of a thermostable firefly luciferase ( tfl ) bearing the peroxisome localization signal to have greater cytoplasmic localization and improved access for its substrate, d-luciferin. Comparison between the three luciferases [ mtfl, tfl , and firefly luciferase ( fl )] both in cell culture and in living mice revealed that mtfl possessed 6- to 10-fold ( in vitro ) and 2-fold ( in vivo ) higher activity than fl . The improved version of the triple fusion vector carrying mtfl as the bioluminescent reporter component showed significantly ( P < 0.05) higher bioluminescence than the previous triple fusion vectors. Of the three different red fluorescent reporter genes ( jred, hcred , and mrfp1 , isolated from jellyfish chromophore, coral Heteractis crispa , and coral Discosoma , respectively) evaluated, mrfp1 was able to preserve highest expression as a component of the triple fusion reporter gene for in vivo fluorescence imaging. A truncated version of wild-type herpes simplex virus 1 (HSV1) thymidine kinase gene ( wttk ) retained a higher expression level than the truncated mutant HSV1-sr39 TK ( ttk ) as the third reporter component of this improved triple fusion vector. Multimodality imaging of tumor-bearing mice using bioluminescence and microPET showed higher luciferase activity [(2.7 ± 0.1 versus 1.9 ± 0.1) × (106 p/s/cm2/sr)] but similar level of fluorine-18–labeled 2′-fluoro-2′-deoxyarabinofuranosyl-5-ethyluracil (18F-FEAU) uptake (1.37 ± 0.15 versus 1.37 ± 0.2) percentage injected dose per gram] by mtfl-mrfp1-wttk –expressing tumors compared with the fl-mrfp1-wttk –expressing tumors. Both tumors showed 4- to 5-fold higher accumulation ( P < 0.05) of 18F-FEAU than fluorine-18–labeled 9-(4-fluoro-3-hydroxymethylbutyl)guanine. This improved triple fusion reporter vector will enable high sensitivity detection of lower numbers of cells from living animals using the combined bioluminescence, fluorescence, and microPET imaging techniques. [Cancer Res 2007;67(7):3085–93]

https://cancerres.aacrjournals.org/content/canres/67/7/3085.full.pdf

Construction and Validation of Improved Triple Fusion Reporter Gene Vectors for Molecular Imaging of Living Subjects

The crystal structures of the full-length Herpes simplex virus type 1 thymidine kinase in its unligated form and in a complex with an adenine analogue have been determined at 1.9 A resolution. The unligated enzyme contains four water molecules in the thymidine pocket and reveals a small induced fit on substrate binding. The structure of the ligated enzyme shows for the first time a bound adenine analogue after numerous complexes with thymine and guanine analogues have been reported. The adenine analogue constitutes a new lead compound for enzyme-prodrug gene therapy. In addition, the structure of mutant Q125N modifying the binding site of the natural substrate thymidine in complex with this substrate has been established at 2.5 A resolution. It reveals that neither the binding mode of thymidine nor the polypeptide backbone conformation is altered, except that the two major hydrogen bonds to thymidine are replaced by a single water-mediated hydrogen bond, which improves the relative acceptance of the prodrugs aciclovir and ganciclovir compared with the natural substrate. Accordingly, the mutant structure represents a first step toward improving the virus-directed enzyme-prodrug gene therapy by enzyme engineering. Proteins 2000;41:545–553. © 2000 Wiley-Liss, Inc.

Nucleoside Binding Site of Herpes Simplex Type 1 Thymidine Kinase Analyzed by X-Ray Crystallography

Kinetic and crystallographic analyses of wild-type Herpes simplex virus type 1 thymidine kinase (TKHSV1) and its Y101F-mutant [TKHSV1(Y101F)] acting on the potent antiviral drug 2‘-exo-methanocarba-thymidine (MCT) have been performed. The kinetic study reveals a 12-fold KM increase for thymidine processed with Y101F as compared to the wild-type TKHSV1. Furthermore, MCT is a substrate for both wild-type and mutant TKHSV1. Its binding affinity for TKHSV1 and TKHSV1(Y101F), expressed as Ki, is 11 μM and 51 μM, respectively, whereas the Ki for human cytosolic thymidine kinase is as high as 1.6 mM, rendering TKHSV1 a selectivity filter for antiviral activity. Moreover, TKHSV1(Y101F) shows a decrease in the quotient of the catalytic efficiency (kcat/KM) of dT over MCT corresponding to an increased specificity for MCT when compared to the wild-type enzyme. Crystal structures of wild-type and mutant TKHSV1 in complex with MCT have been determined to resolutions of 1.7 and 2.4 A, respectively. The thymine moiety o...

Kinetics and crystal structure of the wild-type and the engineered Y101F mutant of Herpes simplex virus type 1 thymidine kinase interacting with (North)-methanocarba-thymidine.

In rational drug design the study of protein-ligand-interactions is one of the most important approaches to get knowledge of SAR. On this study N2-Phenylthioguanines were synthesized by Schiemann reaction from thioguanine followed by a substitution of the fluorine by aniline-derivatives. The activity of these HSV1 TK inhibitors was determined by kinetic measurements of thymidine phosphorylation. The N2-Phenylthioguanines gave the same activity as the oxoanalogues. Interaction energies between thymidine and HSV1 TK were measured by microcalorimetry. Results of the measurement showed negative delta G and delta H values which indicates that the binding of the natural substrate occurs spontaneously and is enthalpy driven.

Modelling of guanine-derivative--protein interaction complexes as a basis of drug design.

Nat. Methods 12, 131–133 (2015); published online 15 December 2014; corrected after print 6 February 2015 In the version of this article initially published, the Hendrickson formula in the Figure 2 legend incorrectly had (2NA/NP)1/2 divided by (f''/Zeff); these terms should have been multiplied. Theerror has been corrected in the HTML and PDF versions of the article.

/pdf/corrigendum-fast-native-sad-phasing-for-routine-1qp7befyhb.pdf

Andrea E. Prota

Papers

Nucleoside Binding Site of Herpes Simplex Type 1 Thymidine Kinase Analyzed by X-Ray Crystallography

Kinetics and crystal structure of the wild-type and the engineered Y101F mutant of Herpes simplex virus type 1 thymidine kinase interacting with (North)-methanocarba-thymidine.

Modelling of guanine-derivative--protein interaction complexes as a basis of drug design.

Corrigendum: Fast native-SAD phasing for routine macromolecular structure determination