NMR Structures of Apo L. casei Dihydrofolate Reductase and Its Complexes with Trimethoprim and NADPH: Contributions to Positive Cooperative Binding from Ligand-Induced Refolding, Conformational Changes, and Interligand Hydrophobic Interactions

bS Supporting Information The enzyme dihydrofolate reductase (DHFR; 5,6,7,8-tetra-hydrofolate:NADPH oxidoreductase, EC 1.5.1.3) catalyzes the reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydro-folate (THF) using NADPH as coenzyme.1 Since THF and its metabolites are precursors of purine and pyrimidine bases, the normal functioning of this enzyme is essential for proliferating cells. This makes DHFR an excellent target for antifolate drugs such as methotrexate (anticancer), pyrimethamine (antimalarial), and trimethoprim (antibacterial). Such agents act by inhibiting the enzyme in parasitic or malignant cells.1,2 The cooperative binding of ligands to DHFR plays an important role not only in the enzyme catalytic cycle (negative cooperativity in THF/ NADPH binding)3 but also in enzyme inhibition (positive cooperativity in antifolate/NADPH binding).4 The effects of positive cooperative binding in controlling enzyme inhibition ar

Computer-assisted methods for molecular structure elucidation: realizing a spectroscopist's dream

Abstract Background This article coincides with the 40 year anniversary of the first published works devoted to the creation of algorithms for computer-aided structure elucidation (CASE). The general principles on which CASE methods are based will be reviewed and the present state of the art in this field will be described using, as an example, the expert system Structure Elucidator. Results The developers of CASE systems have been forced to overcome many obstacles hindering the development of a software application capable of drastically reducing the time and effort required to determine the structures of newly isolated organic compounds. Large complex molecules of up to 100 or more skeletal atoms with topological peculiarity can be quickly identified using the expert system Structure Elucidator based on spectral data. Logical analysis of 2D NMR data frequently allows for the detection of the presence of COSY and HMBC correlations of "nonstandard" length. Fuzzy structure generation provides a possibility to obtain the correct solution even in those cases when an unknown number of nonstandard correlations of unknown length are present in the spectra. The relative stereochemistry of big rigid molecules containing many stereocenters can be determined using the StrucEluc system and NOESY/ROESY 2D NMR data for this purpose. Conclusion The StrucEluc system continues to be developed in order to expand the general applicability, provide improved workflows, usability of the system and increased reliability of the results. It is expected that expert systems similar to that described in this paper will receive increasing acceptance in the next decade and will ultimately be integrated directly to analytical instruments for the purpose of organic analysis. Work in this direction is in progress. In spite of the fact that many difficulties have already been overcome to deliver on the spectroscopist's dream of "fully automated structure elucidation" there is still work to do. Nevertheless, as the efficiency of expert systems is enhanced the solution of increasingly complex structural problems will be achievable.</p

CRISPR Mediated Target Validation of ERCC3 as the Target of Triptolide

Exome sequencing of Triptolide-resistant haploid cells identified mutations in TFIIH complex, with a majority of them in the ERCC3 subunit. These mutations turned out to be recessive, therefore difficult to validate using conventional cDNA overexpression approach. Here we adopted a novel CRISPR-based knock-in strategy which makes it very straightforward to re-introduce the recessive point mutations that confer drug resistance phenotype in cells. Coupled with mutagenesis and next generation sequencing, this novel gene-editing strategy can expedite small molecule target identification and validation in mammalian cells

Empirical and DFT GIAO quantum-mechanical methods of 13C chemical shifts prediction: competitors or collaborators?

<p>The accuracy of 13C chemical shift prediction by both DFT GIAO quantum-mechanical (QM) and empirical methods was compared using 205 structures for which experimental and QM-calculated chemical shifts were published in the literature. For these structures, 13C chemical shifts were calculated using HOSE code and neural network (NN) algorithms developed within our laboratory. In total, 2531 chemical shifts were analyzed and statistically processed. It has been shown that, in general, QM methods are capable of providing similar but inferior accuracy to the empirical approaches, but quite frequently they give larger mean average error values. For the structural set examined in this work, the following mean absolute errors (MAEs) were found: MAE(HOSE) = 1.58 ppm, MAE(NN) = 1.91 ppm and MAE(QM) = 3.29 ppm. A strategy of combined application of both the empirical and DFT GIAO approaches is suggested. The strategy could provide a synergistic effect if the advantages intrinsic to each method are exploited.</p

Computer-Assisted Methods for Molecular Structure Elucidation: Realizing a Spectroscopist′s Dream

<p>This article coincides with the 40 year anniversary of the first published works devoted to the creation of algorithms for computer-aided structure elucidation (CASE). The general principles on which CASE methods are based will be reviewed and the present state of the art in this field will be described using, as an example, the expert system Structure Elucidator.</p

Staurosporine Increases Lentiviral Vector Transduction Efficiency of Human Hematopoietic Stem and Progenitor Cells

Lentiviral vector (LVV)-mediated transduction of human CD34+ hematopoietic stem and progenitor cells (HSPCs) holds tremendous promise for the treatment of monogenic hematological diseases. This approach requires the generation of a sufficient proportion of gene-modified cells. We identified staurosporine, a serine/threonine kinase inhibitor, as a small molecule that could be added to the transduction process to increase the proportion of genetically modified HSPCs by overcoming a LVV entry barrier. Staurosporine increased vector copy number (VCN) approximately 2-fold when added to mobilized peripheral blood (mPB) CD34+ cells prior to transduction. Limited staurosporine treatment did not affect viability of cells post-transduction, and there was no difference in in vitro colony formation compared to vehicle-treated cells. Xenotransplantation studies identified a statistically significant increase in VCN in engrafted human cells in mouse bone marrow at 4 months post-transplantation compared to vehicle-treated cells. Prostaglandin E2 (PGE2) is known to increase transduction efficiency of HSPCs through a different mechanism. Combining staurosporine and PGE2 resulted in further enhancement of transduction efficiency, particularly in short-term HSPCs. The combinatorial use of small molecules, such as staurosporine and PGE2, to enhance LVV transduction of human CD34+ cells is a promising method to improve transduction efficiency and subsequent potential therapeutic benefit of gene therapy drug products. Keywords: lentiviral, HSPC, transductio

NMR Structures of Apo <i>L. casei</i> Dihydrofolate Reductase and Its Complexes with Trimethoprim and NADPH: Contributions to Positive Cooperative Binding from Ligand-Induced Refolding, Conformational Changes, and Interligand Hydrophobic Interactions

In order to examine the origins of the large positive cooperativity (Δ<i>G</i>₀^coop = −2.9 kcal mol⁻¹) of trimethoprim (TMP) binding to a bacterial dihydrofolate reductase (DHFR) in the presence of NADPH, we have determined and compared NMR solution structures of <i>L. casei</i> apo DHFR and its binary and ternary complexes with TMP and NADPH and made complementary thermodynamic measurements. The DHFR structures are generally very similar except for the A−B loop region and part of helix B (residues 15−31) which could not be directly detected for <i>L. casei</i> apo DHFR because of line broadening from exchange between folded and unfolded forms. Thermodynamic and NMR measurements suggested that a significant contribution to the cooperativity comes from refolding of apo DHFR on binding the first ligand (up to −0.95 kcals mol⁻¹ if 80% of A−B loop requires refolding). Comparisons of Cα−Cα distance differences and domain rotation angles between apo DHFR and its complexes indicated that generally similar conformational changes involving domain movements accompany formation of the binary complexes with either TMP or NADPH and that the binary structures are approaching that of the ternary complex as would be expected for positive cooperativity. These favorable ligand-induced structural changes upon binding the first ligand will also contribute significantly to the cooperative binding. A further substantial contribution to cooperative binding results from the proximity of the bound ligands in the ternary complex: this reduces the solvent accessible area of the ligand and provides a favorable entropic hydrophobic contribution (up to −1.4 kcal mol⁻¹)

DNA sequencing and CRISPR-Cas9 gene editing for target validation in mammalian cells

Identification and validation of drug-resistant mutations can provide important insights into the mechanism of action of a compound. Here we demonstrate the feasibility of such an approach in mammalian cells using next-generation sequencing of drug-resistant clones and CRISPR-Cas9-mediated gene editing on two drug-target pairs, 6-thioguanine-HPRT1 and triptolide-ERCC3. We showed that disrupting functional HPRT1 allele or introducing ERCC3 point mutations by gene editing can confer drug resistance in cell