Discovery of a natural product that binds to the mycobacterium tuberculosis protein Rv1466 using native mass spectrometry

Elucidation of the mechanism of action of compounds with cellular bioactivity is important for progressing compounds into future drug development. In recent years, phenotype-based drug discovery has become the dominant approach to drug discovery over target-based drug discovery, which relies on the knowledge of a specific drug target of a disease. Still, when targeting an infectious disease via a high throughput phenotypic assay it is highly advantageous to identifying the compound’s cellular activity. A fraction derived from the plant Polyalthia sp. showed activity against Mycobacterium tuberculosis at 62.5 µge/µL. A known compound, altholactone, was identified from this fraction that showed activity towards M. tuberculosis at an minimum inhibitory concentration (MIC) of 64 µM. Retrospective analysis of a target-based screen against a TB proteome panel using native mass spectrometry established that the active fraction was bound to the mycobacterial protein Rv1466 with an estimated pseudo-Kd of 42.0 ± 6.1 µM. Our findings established Rv1466 as the potential molecular target of altholactone, which is responsible for the observed in vivo toxicity towards M. tuberculosis

Amelogenin Supramolecular Assembly in Nanospheres Defined by a Complex Helix-Coil-PPII Helix 3D-Structure

Tooth enamel, the hardest material in the human body, is formed within a self-assembled matrix consisting mostly of amelogenin proteins. Here we have determined the complete mouse amelogenin structure under physiological conditions and defined interactions between individual domains. NMR spectroscopy revealed four major amelogenin structural motifs, including an N-terminal assembly of four α-helical segments (S9-V19, T21-P33, Y39-W45, V53-Q56), an elongated random coil region interrupted by two 310 helices (∼P60-Q117), an extended proline-rich PPII-helical region (P118-L165), and a charged hydrophilic C-terminus (L165-D180). HSQC experiments demonstrated ipsilateral interactions between terminal domains of individual amelogenin molecules, i.e. N-terminal interactions with corresponding N-termini and C-terminal interactions with corresponding C-termini, while the central random coil domain did not engage in interactions. Our HSQC spectra of the full-length amelogenin central domain region completely overlapped with spectra of the monomeric Amel-M fragment, suggesting that the central amelogenin coil region did not involve in assembly, even in assembled nanospheres. This finding was confirmed by analytical ultracentrifugation experiments. We conclude that under conditions resembling those found in the developing enamel protein matrix, amelogenin molecules form complex 3D-structures with N-terminal α-helix-like segments and C-terminal PPII-helices, which self-assemble through ipsilateral interactions at the N-terminus of the molecule

SAD phasing using iodide ions in a high-throughput structural genomics environment

The Seattle Structural Genomics Center for Infectious Disease (SSGCID) focuses on the structure elucidation of potential drug targets from class A, B, and C infectious disease organisms. Many SSGCID targets are selected because they have homologs in other organisms that are validated drug targets with known structures. Thus, many SSGCID targets are expected to be solved by molecular replacement (MR), and reflective of this, all proteins are expressed in native form. However, many community request targets do not have homologs with known structures and not all internally selected targets readily solve by MR, necessitating experimental phase determination. We have adopted the use of iodide ion soaks and single wavelength anomalous dispersion (SAD) experiments as our primary method for de novo phasing. This method uses existing native crystals and in house data collection, resulting in rapid, low cost structure determination. Iodide ions are non-toxic and soluble at molar concentrations, facilitating binding at numerous hydrophobic or positively charged sites. We have used this technique across a wide range of crystallization conditions with successful structure determination in 16 of 17 cases within the first year of use (94% success rate). Here we present a general overview of this method as well as several examples including SAD phasing of proteins with novel folds and the combined use of SAD and MR for targets with weak MR solutions. These cases highlight the straightforward and powerful method of iodide ion SAD phasing in a high-throughput structural genomics environment

Justice: Greater Access, Lower Costs

Litigation imposes large costs on society; this justifies settlement considerations. In any case, access to justice is critical to socioeconomic development; as such, it needs to be balanced with litigation minimization. This study examines the tradeoff between litigation and access to justice and explicitly elucidates their relationship. In considering access issues, this study finds that the outcomes of policies that affect parties’ litigation decisions partially depart from those in the standard literature. For instance, increasing parties’ litigation costs does not necessarily promote settlement in the shadow of the court. Rather, effects depend on the elasticity of the demand for legal remedies. Furthermore, even while pushing litigation, enhancing access to justice is efficient as long as the claimant’s marginal propensity to litigate is smaller than the social opportunity-cost of access to justice. This finding offers further insight into the suitability of litigation subsidization through legal aid

Influence of nucleic acid base aromaticity on substrate reactivity with enzymes acting on single-stranded DNA.

Stacking between aromatic amino acids and nucleic acid bases may play an important role in the interaction of enzymes with nucleic acid substrates. In such circumstances, disruption of base aromaticity would be expected to decrease enzyme activity on the modified substrates. We have examined the requirement for DNA base aromaticity of five enzymes that act on single-stranded DNA, T4 polynucleotide kinase, nucleases P1 and S1, and snake venom and calf spleen phosphodiesterases, by comparing their kinetics of reaction with a series of dinucleoside monophosphates containing thymidine or a ring-saturated derivative. The modified substrates contained either cis-5R,6S-di-hydro-5,6-dihydroxythymidine (thymidine glycol) or a mixture of the 5R and 5S isomers of 5,6-dihydrothymidine. It was observed that for all the enzymes, except snake venom phosphodiesterase, the parent molecules were better substrates than the dihydrothymidine derivatives, while the thymidine glycol compounds were significantly poorer substrates. Snake venom phosphodiesterase acted on the unmodified and dihydrothymidine molecules at almost the same rate. These results imply that for all the remaining enzymes base aromaticity is a factor in enzyme-substrate interaction, but that additional factors must contribute to the poorer substrate capacity of the thymidine glycol compounds. The influence of the stereochemistry of the dihydrothymidine derivatives was also investigated. We observed that nuclease P1 and S1 hydrolysed the molecules containing 5R-dihydrothymidine approximately 50-times faster than those containing the S-isomer. The other enzymes displayed no measurable stereospecificity

Photooxidation of d(TpG) by phthalocyanines and riboflavin. Isolation and characterization of dinucleoside monophosphates containing the 4R* and 4S* diastereoisomers of 4,8-dihydro-4-hydroxy-8-oxo-2'-deoxy-guanosine.

Phthalocyanine mediated photosensitization of 2'-deoxyguanosine (dG) in oxygen saturated aqueous solution has previously been shown to result in the addition of molecular oxygen to the guanine base generating the 4R* and 4S* diastereoisomers of 4,8-dihydro-4-hydroxy-8-oxo-2'-deoxyguanosine (dO) (the asterisk denotes unambiguous assignment of the 4R and 4S diastereoisomers). The data presented here show that the same guanine modified bases are generated in a 1:1 ratio when thymidylyl-(3',5')-2'-deoxyguanosine (d(TpG)) is similarly photo-oxidized. These modified dinucleoside monophosphates, labelled d(TpO)-A and -B, have been isolated by high performance liquid chromatography and characterized by proton NMR spectrometry, fast atom bombardment mass spectrometry, and enzymatic digestions. Photosensitization in D2O instead of H2O leads to an increase in the rate of d(TpO) formation that is consistent with a type II (singlet oxygen) reaction mechanism. Three interesting properties of these modified dinucleoside monophosphates are: i) the rate of their digestion with spleen phosphodiesterase is greatly reduced relative to d(TpG), ii) they are not digested by snake venom phosphodiesterase, and iii) they are stable to 1.0 M piperidine at 90 degrees C for 30 min. The latter observation indicates that 4,8-dihydro-4-hydroxy-8-oxoguanine is not a base lesion responsible for the strand breaks observed following hot piperidine treatment of DNA exposed to type II photosensitizers or chemically generated singlet oxygen

Infrared spectroscopy of human apolipoprotein fragments in SDS/D2O :\u2009 relative lipid-binding affinities and a novel amide I assignment

Infrared absorption spectra are reported for six apolipoprotein fragments in SDS/D2O. Five of the peptides correspond to proposed lipid-binding domains of human apolipoproteins [apoC-I(7 1224), apoC-I(35 1253), apoA-II(18 1230)+, apoA-I(166 12185), apoE(267 12289)], and the sixth is the de novo lipid associating peptide LAP-20. The amide I infrared absorption patterns are generally consistent with predominantly helical structures (as determined previously by NMR spectroscopy and distance geometry calculations) and further suggest that apoA-I(166 12185) and apoE(267 12289) are bound to SDS relatively weakly in comparison to the other four peptides. The latter conclusion is also supported by the temperature dependence of the infrared spectra, as increasing temperature promotes a distinct increase in random coil structure only for apoA-I(166 12185) and apoE(267 12289). In addition to features readily ascribed to helices, the infrared spectra of all the peptides show absorptions in the spectral region 1630 121635 cm-1 that is usually associated with \u3b2-structure, a motif that is clearly absent from the NMR-derived structures. Parallel difficulties also arose in the analyses of the circular dichroism spectra. We suggest that both the low-frequency infrared absorptions and the ambiguities in interpreting the CD spectra may be due to unusual structures at the peptide C-termini, involving CO groups that form hydrogen bonds simultaneously either with two solvent molecules or with donors from the backbone (NH) and the solvent (OH). Analogous absorptions may be a general feature of solvent-exposed helices, which suggests a need for caution in assigning amide I bands below 1640 cm-1.Peer reviewed: YesNRC publication: Ye