Edward R. Zartler

Practical aspects of NMR-based fragment discovery.

Fragment-based drug discovery (FBDD) needs a biophysical assay to complement, or even replace, biochemical screening. NMR is the best choice for this because NMR delivers many different types of data that impacts medicinal chemistry decisions. There are a multitude of different NMR methods which can be employed to these ends. The choice of which method to use will be different for every need. We discuss the different methods, the data they produce, and how they are best utilized in a FBDD setting.

1D NMR Methods in Ligand-Receptor Interactions

The drug discovery process often involves the screening of compound libraries to identify drug candidates capable of binding to target macromolecules. New approaches in biological and chemical research are driving a change in the pharmaceutical industry. Recent advances in NMR spectroscopy such as affinity NMR techniques, which detect binding of a small molecule with a “receptor”, have been shown to be valuable tools to perform rapid screening of compounds for biological activity. These NMR observable events include using relaxation, chemical shift perturbations, translational diffusion, and magnetization transfer. These one dimensional NMR methods increase both the throughput of screening and yield crucial data on the mode of binding. The practical utility of these techniques will be described.

Structure of the capsular polysaccharide of pneumococcal serotype 11A reveals a novel acetylglycerol that is the structural basis for 11A subtypes.

We have undertaken a structural assessment of Streptococcus pneumoniae 11A polysaccharide as well as two clinical isolates related to 11A. The clinical isolates were labeled 11Aα and 11Aβ. The result of our experiments is a revision to the old structure for S. pneumoniae 11A polysaccharide. The new structure differs from the old structure in both the primary connectivities and acetylation pattern. We also show that 11A contains an acetylglycerol-PO4 moiety, a substitution that is heretofore unknown in the bacterial polysaccharide literature. The two clinical isolates were also structurally characterized. 11Aα was determined to be identical to 11A. 11Aβ is a new serotype, which differs from 11A in the absence of the acetylation of the glycerol-PO4 moiety and a different acetylation pattern of the saccharides. Thus, we propose that the acetylglycerol is the structural basis for 11Aα and 11Aβ subtypes.

Streptococcus pneumoniae Serotype 11D Has a Bispecific Glycosyltransferase and Expresses Two Different Capsular Polysaccharide Repeating Units

Background: Streptococcus pneumoniae serotype 11D capsular polysaccharide (CPS) structure is unknown. Results: Serotype 11D PS contains two different repeating units; one has αGlcNAc, and the other contains αGlc. Conclusion: The 11D CPS is due to the bispecific glycosyltransferase WcrL. Based on codon 112, WcrL can transfer αGlc, αGlcNAc, or both. Significance: Minimal genetic changes can make bacteria produce different polysaccharides. Streptococcus pneumoniae (pneumococcus) expresses a capsular polysaccharide (CPS) that protects against host immunity and is synthesized by enzymes in the capsular polysaccharide synthesis (cps) locus. Serogroup 11 has six members (11A to -E) and the CPS structure of all members has been solved, except for serotype 11D. The cps loci of 11A and 11D differ by one codon (N112S) in wcrL, which putatively encodes a glycosyltransferase that adds the fourth sugar of the CPS repeating unit (RU). Gas chromatography and nuclear magnetic resonance analysis revealed that 11A and 11D PSs contain identical CPS RUs that contain αGlc as the fourth sugar. However, ∼25% of 11D CPS RUs contain instead αGlcNAc as the fourth sugar, suggesting that 11D wcrL encodes a bispecific glycosyltransferase. To test the hypothesis that codon 112 of WcrL determines enzyme specificity, and therefore the fourth sugar in the RU, we generated three isogenic pneumococcal strains with 11A cps loci containing wcrL encoding Ser-112 (MBO128) or Ala-112 (MBO130). MBO128 was serologically and biochemically identical to serotype 11D. MBO130 has a unique serologic profile; has as much αGlcNAc as 11F, 11B, and 11C CPS do; and may represent a new serotype. These findings demonstrate how pneumococci alter their CPS structure and their immunologic properties with a minimal genetic change.

Elucidation of structural and antigenic properties of pneumococcal serotype 11A, 11B, 11C, and 11F polysaccharide capsules.

Despite the emerging impact of serogroup 11 serotypes in Streptococcus pneumoniae epidemiology, the structures of serogroup 11 capsule types have not been fully elucidated, particularly the locations of O-acetyl substitutions. Here, we report the complete structures of the serotype 11B, 11C, and 11F polysaccharides and a revision to the serotype 11A capsular polysaccharide using nuclear magnetic resonance (NMR). All structures shared a linear, tetrasaccharide backbone with a pendant phosphopolyalcohol. Three of four saccharides are conserved in all serotypes. The individual serotype capsules differed in the identity of one saccharide, the pendant phosphopolyalcohol, and the O-acetylation pattern. Though the assigned locations of O-acetate substitutions in this study differed from those of previous reports, our findings were corroborated with strong correlations to serology and genetics. We examined the binding of serotyping sera to serogroup 11 polysaccharides by using flow cytometry and an inhibition-type enzyme-linked immunosorbent assay (ELISA) and found that de-O-acetylation of capsular polysaccharides by mild hydrolysis decreases its immunoreactivity, supporting the crucial role of O-acetylation in the antigenicity of these polysaccharides. Due to strong correlations between polysaccharide structures and capsule biosynthesis genes, we were able to assign target substrates for the O-acetyltransferases encoded by wcwC, wcwR, wcwT, and wcjE. We identified antigenic determinants for serogroup 11 serotyping sera and highlight the idea that conventional serotyping methods are not capable of recognizing all putative variants of S. pneumoniae serogroup 11.

The use of 1H–31P GHMBC and covariance NMR to unambiguously determine phosphate ester linkages in complex polysaccharide mixtures

Poly- and oligo-saccharides are commonly employed as antigens in many vaccines. These antigens contain phosphoester structural elements that are crucial to the antigenicity, and hence the effectiveness of the vaccine. Nuclear Magnetic Resonance (NMR) is a powerful tool for the site-specific identification of phosphoesters in saccharides. We describe here two advances in the characterization of phosphoesters in saccharides: (1) the use of 1H–31P GHMBC to determine the site-specific identity of phosphoester moieties in heterogeneous mixtures and (2) the use of Unsymmetrical/Generalized Indirect Covariance (U/GIC) to calculate a carbon-phosphorus 2D spectrum. The sensitivity of the 1H–31P GHMBC is far greater than the “standard” 1H–31P GHSQC and allows long-range 3–5JHP couplings to be readily detected. This is the first example to be reported of using U/GIC to calculate a carbon-phosphorus spectrum. The U/GIC processing affords, in many cases, a fivefold to tenfold or greater increase in signal-to-noise ratios in the calculated spectrum. When coupled with the high sensitivity of 1H–31P HMBC, U/GIC processing allows the complete and unambiguous assignments of phosphoester moieties present in heterogeneous samples at levels of ~5% (or less) of the total sample, expanding the breadth of samples that NMR can be used to analyze. This new analytical technique is generally applicable to any NMR-observable phosphoester.

Identification of 3-O-acetylglycerol, a novel structural element in bacterial polysaccharides.

We have discovered a novel bacterial polysaccharide structural element, 3-O-acetylglycerol, in the Streptococcus pneumoniae ST11A polysaccharide: This moiety was elucidated through a combination of homonuclear and heteronuclear 1D and 2D NMR experiments using (1)H, (13)C, and (31)P in various combinations. The 3-O-acetylglycerol moiety is substoichiometrically O-acetylated in ST11A; yet, key connectivities that unequivocally show O-acetylation at the glycerol are provided by the long-range correlations from the acetate methyl groups to the glycerol in the (1)H-(13)C HMBC spectrum. Additionally, we clarify the (1)H-(31)P assignments previously presented.