Prostaglandin E2 Increases Lentiviral Vector Transduction Efficiency of Adult Human Hematopoietic Stem and Progenitor Cells.

Gene therapy currently in development for hemoglobinopathies utilizes ex vivo lentiviral transduction of CD34+ hematopoietic stem and progenitor cells (HSPCs). A small-molecule screen identified prostaglandin E2 (PGE2) as a positive mediator of lentiviral transduction of CD34+ cells. Supplementation with PGE2 increased lentiviral vector (LVV) transduction of CD34+ cells approximately 2-fold compared to control transduction methods with no effect on cell viability. Transduction efficiency was consistently increased in primary CD34+ cells from multiple normal human donors and from patients with ß-thalassemia or sickle cell disease. Notably, PGE2 increased transduction of repopulating human HSPCs in an immune-deficient (nonobese diabetic/severe combined immunodeficiency/interleukin-2 gamma receptor null [NSG]) xenotransplantation mouse model without evidence of in vivo toxicity, lineage bias, or a de novo bias of lentiviral integration sites. These data suggest that PGE2 improves lentiviral transduction and increases vector copy number, therefore resulting in increased transgene expression. As a result, PGE2 may be useful in clinical gene therapy applications using lentivirally modified HSPCs.

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 cells.

Small molecules discovered in a pathway screen target the Rho pathway in cytokinesis.

We report the discovery of small molecules that target the Rho pathway, which is a central regulator of cytokinesis--the final step in cell division. We have developed a way of targeting a small molecule screen toward a specific pathway, which should be widely applicable to the investigation of any signaling pathway. In a chemical genetic variant of a classical modifier screen, we used RNA interference (RNAi) to sensitize cells and identified small molecules that suppressed or enhanced the RNAi phenotype. We discovered promising candidate molecules, which we named Rhodblock, and we identified the target of Rhodblock as Rho kinase. Several Rhodblocks inhibited one function of the Rho pathway in cells: the correct localization of phosphorylated myosin light chain during cytokinesis. Rhodblocks differentially perturb Rho pathway proteins in cells and can be used to dissect the mechanism of the Rho pathway during cytokinesis.

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.

In order to examine the origins of the large positive cooperativity (ΔG(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 L. casei 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 L. casei 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)).

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

The accuracy of (13)C 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, (13)C 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.

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.

Binucleine 2, an isoform-specific inhibitor of Drosophila Aurora B kinase, provides insights into the mechanism of cytokinesis.

Aurora kinases are key regulators of cell division and important targets for cancer therapy. We report that Binucleine 2 is a highly isoform-specific inhibitor of Drosophila Aurora B kinase, and we identify a single residue within the kinase active site that confers specificity for Aurora B. Using Binucleine 2, we show that Aurora B kinase activity is not required during contractile ring ingression, providing insight into the mechanism of cytokinesis.

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

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.

Toward more reliable 13C and 1H chemical shift prediction: a systematic comparison of neural-network and least-squares regression based approaches.

The efficacy of neural network (NN) and partial least-squares (PLS) methods is compared for the prediction of NMR chemical shifts for both 1H and 13C nuclei using very large databases containing millions of chemical shifts. The chemical structure description scheme used in this work is based on individual atoms rather than functional groups. The performances of each of the methods were optimized in a systematic manner described in this work. Both of the methods, least-squares and neural network analyses, produce results of a very similar quality, but the least-squares algorithm is approximately 2--3 times faster.

Calculating protein structures directly from anisotropic spin interaction constraints.

Protein structure determination by solid-state NMR of aligned samples relies on the fundamental characteristics of the anisotropic nuclear spin interactions present in isotopically labeled proteins. Progress in the implementation of algorithms that calculate protein structures from the orientational constraints in the chemical shift and heteronuclear dipolar coupling interactions is described using both simulated and experimental data.

Exceptionally facile CO addition to a saturated ruthenium complex.

Synthesis and characterization of Cp*Ru[eta3-HC(PPh2NPh)2], 1, reveals it to have a "piano stool" structure with the ligand bound to Ru(II) via two N and the unique, sp3 hybridized carbon. While the analogous (cymene) Ru[eta3-HC(PPh2NPh)2]+ does not react with CO, under the same conditions, 1 adds one CO rapidly (25 degrees C, 1 atm CO). Characterization, including an X-ray structure determination, shows that CO has displaced one chelate ligand nitrogen, which then hangs off the molecule, free of Ru. DFT calculations reveal a possible mechanism via a remarkably low energy (+9.3 kcal/mol) intermediate, pendant N, but with one phenyl on phosphorus stabilizing Ru via donation from a C(ipso)=C(ortho) bond. DFT calculations show that the electronic energy change for binding CO is over 20 kcal/mol less favorable for cymene than for C5Me5- as ligand; the reactivity difference is thus thermodynamic in origin.

Multifunctional behavior by a Bis-(phosphinimino)methanide ligand: eta 2- vs eta 3-coordination vs Bronsted basicity.

The reaction of [(Cymene)RuCl2]2 with the chelate LiHC(PPh2NPh)2 occurs to remove both chloride ligands, to furnish a cationic Ru(II) complex with the monoanionic ligand bound eta3, through two N and an sp3 carbon. This cation is also produced from the conjugate acid of the ligand H2C(PPh2NPh)2 because this molecule can serve as a Brønsted base, to deprotonate the acidic carbon of another molecule of H2C(PPh2NPh)2. DFT calculations show an energy surface where (Cymene)RuHC(PPh2NPh)2L is more stable with a Ru-CH(PPh2NPh)2 bond and with L = Cl- or MeCN not coordinated to Ru, than to an eta2-HC(PPh2NPh)2 structure with coordinated L; this is tested experimentally. The greater tendency for this ligand to be coordinated eta3 vs analogous diketiminates is discussed. The nucleophilicity of Cgamma in structure 1, vs that of donors L = Cl- or MeCN, is evaluated to understand the preference of the bis(phosphinimino)methanide to be bidentate or tridentate.