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Biomedical subjects

Krishna Saxena

Publications and source records attributed to Krishna Saxena.

At least 19 recordsLinked to original sources
PubMed2006

Brunsvicamides A-C: sponge-related cyanobacterial peptides with Mycobacterium tuberculosis protein tyrosine phosphatase inhibitory activity.

The cyanobacterium Tychonema sp. produces the new cyclic hexapeptides brunsvicamide A-C (1-3). Brunsvicamide B (2) and C (3) selectively inhibit the Mycobacterium tuberculosis protein tyrosine phosphatase B (MptpB), a potential drug target for tuberculosis therapy for which no inhibitors are known to date. Brunsvicamide C contains an N-methylated N'-formylkynurenine moiety, a unique structural motif in cyclic peptides. The new peptides are related to the sponge-derived mozamides, supporting the suggestion that secondary metabolites of certain marine invertebrates are produced by associated microorganisms. Thus, microorganisms phylogenetically related to symbionts of marine invertebrates can be judged as a means to supply "marine-like" compounds for drug development.

Animals↗
PubMed2006

Discovery of protein phosphatase inhibitor classes by biology-oriented synthesis.

Protein phosphatases have very recently emerged as important targets for chemical biology and medicinal chemistry research, and new phosphatase inhibitor classes are in high demand. The underlying frameworks of natural products represent the evolutionarily selected fractions of chemical space explored by nature so far and meet the criteria of relevance to nature and biological prevalidation most crucial to inhibitor development. We refer to synthesis efforts and compound collection development based on these criteria as biology-oriented synthesis. For the discovery of phosphatase inhibitor classes by means of this approach, four natural product-derived or -inspired medium-sized compound collections were synthesized and investigated for inhibition of the tyrosine phosphatases VE-PTP, Shp-2, PTP1B, MptpA, and MptpB and the dual-specificity phosphatases Cdc25A and VHR. The screen yielded four unprecedented and selective phosphatase inhibitor classes for four phosphatases with high hit rates. For VE-PTP and MptpB the first inhibitors were discovered. These results demonstrate that biology-oriented synthesis is an efficient approach to the discovery of new compound classes for medicinal chemistry and chemical biology research that opens up new opportunities for the study of phosphatases, which may lead to the development of new drug candidates.

Biological Products↗
PubMed2006

Biomolecular NMR: a chaperone to drug discovery.

Biomolecular NMR now contributes routinely to every step in the development of new chemical entities ahead of clinical trials. The versatility of NMR--from detection of ligand binding over a wide range of affinities and a wide range of drug targets with its wealth of molecular information, to metabolomic profiling, both ex vivo and in vivo--has paved the way for broadly distributed applications in academia and the pharmaceutical industry. Proteomics and initial target selection both benefit from NMR: screenings by NMR identify lead compounds capable of inhibiting protein-protein interactions, still one of the most difficult development tasks in drug discovery. NMR hardware improvements have given access to the microgram domain of phytochemistry, which should lead to the discovery of novel bioactive natural compounds. Steering medicinal chemists through the lead optimisation process by providing detailed information about protein-ligand interactions has led to impressive success in the development of novel drugs. The study of biofluid composition--metabonomics--provides information about pharmacokinetics and helps toxicological safety assessment in animal model systems. In vivo, magnetic resonance spectroscopy interrogates metabolite distributions in living cells and tissues with increasing precision, which significantly impacts the development of anticancer or neurological disorder therapeutics. An overview of different steps in recent drug discovery is presented to illuminate the links with the most recent advances in NMR methodology.

Drug Design↗
PubMed2006

NMR analysis of a Tau phosphorylation pattern.

The phosphorylation of the neuronal Tau protein modulates both its physiological role of microtubule binding and its aggregation into paired helical fragments observed in Alzheimer's diseased neurons. However, detailed knowledge of the role of phosphorylation at specific sites has been hampered by the analytical difficulties to evaluate the level of site-specific phosphate incorporation. Even with recombinant kinases, mass spectrometry and immunodetection are not evident for determining the full phosphorylation pattern in a qualitative and quantitative manner. We show here that heteronuclear NMR spectroscopy on a 15N labeled Tau sample modified by the cAMP dependent kinase allows identification of all phosphorylation sites, measures their level of phosphate integration, and yields kinetic data for the enzymatic modification of the individual sites. Filtering through the 15N label discards the necessity of any further sample purification and allows the in situ monitoring of kinase activity at selected sites. We finally demonstrate that the NMR approach can equally be used to evaluate potential kinase inhibitors in a straightforward manner.

Adenosine Triphosphate↗
PubMed2005

Polo-like kinase 1-mediated phosphorylation stabilizes Pin1 by inhibiting its ubiquitination in human cells.

The Polo-like kinase 1 (Plk1) is a key regulator of mitosis. It is reported that the human peptidyl-prolyl cis/trans-isomerase Pin1 binds to Plk1 from mitotic cell extracts in vitro. Here we demonstrate that Ser-65 in Pin1 is the major site for Plk1-specific phosphorylation, and the polo-box domain of Plk1 is required for this phosphorylation. Interestingly, the phosphorylation of Pin1 by Plk1 does not affect its isomerase activity but rather is linked to its protein stability. Pin1 is ubiquitinated in HeLa S3 cells, and substitution of Glu for Ser-65 reduces the ubiquitination of Pin1. Furthermore, inhibition of Plk1 activity by expression of a dominant negative form of Plk1 or by transfection of small interfering RNA targeted to Plk1 enhances the ubiquitination of Pin1 and subsequently reduces the amount of Pin1 in human cancer cells. Since previous reports suggested that Plk1 is a substrate of Pin1, our work adds a new dimension to this interaction of two important mitotic regulators.

Blotting, Western↗
PubMed2005

Folding and activity of cAMP-dependent protein kinase mutants.

The catalytic subunit of cAMP-dependent protein kinase (PKA) can easily be expressed in Escherichia coli and is catalytically active. Four phosphorylation sites are known in PKA (S10, S139, T197 and S338), and the isolated recombinant protein is a mixture of different phosphorylated forms. Obtaining uniformly phosphorylated protein requires separation of the protein preparation leading to significant loss in protein yield. It is found that the mutant S10A/S139D/S338D has similar properties as the wild-type protein, whereas additional replacement of T197 with either E or D reduces protein expression yield as well as folding propensity of the protein. Due to its high sequence homology to Akt/PKB, which cannot easily be expressed in E. coli, PKA has been used as a surrogate kinase for drug design. Several mutations within the ATP binding site have been described to make PKA even more similar to Akt/PKB. Two proteins with Akt/PKB-like mutations in the ATP binding site were made (PKAB6 and PKAB8), and in addition S10, S139 and S338 phosphorylation sites have been removed. These proteins can be expressed in high yields but have reduced activity compared to the wild-type. Proper folding of all proteins was analyzed by 2D 1H, 15N-TROSY NMR experiments.

Cyclic AMP-Dependent Protein Kinases↗
PubMed2005

QSAR-by-NMR: quantitative insights into structural determinants for binding affinity by analysis of 1H/15N chemical shift differences in MMP-3 ligands.

A novel strategy is applied to obtain quantitative insights on factors influencing biological affinity in protein-ligand complexes. This approach is based on the detection of ligand binding by (15)N and (1)H amide chemical shift differences in two-dimensional (15)N-heteronuclear single-quantum correlation spectra. Essential structural features linked to affinity can be extracted using statistical analysis of (15)N and (1)H amide chemical shift differences in congeneric series relative to uncomplexed protein spectra, as demonstrated for 20 MMP-3 inhibitors in complex with human matrix metalloproteinase stromelysin (MMP-3). The statistical analysis using PLS led to a significant model, while its chemical interpretation, highlighting the importance of particular residues for affinity, are in agreement to an X-ray structure of one key compound in the homologue MMP-8 binding site.

Amides↗
PubMed2005

How much NMR data is required to determine a protein-ligand complex structure?

Here we present an NMR-based approach to solving protein-ligand structures. The procedure is guided by biophysical, biochemical, or knowledge-based data. The structures are mainly derived from ligand-induced chemical-shift perturbations (CSP) induced in the resonances of the protein and ligand-detected saturated transfer difference signals between ligands and selectively labeled proteins (SOS-NMR). Accuracy, as judged by comparison with X-ray results, depends on the nature and completeness of the experimental data. An experimental protocol is proposed that starts with calculations that make use of readily available chemical-shift perturbations as experimental constraints. If necessary, more sophisticated experimental results have to be added to improve the accuracy of the protein-ligand complex structure. The criteria for evaluation and selection of meaningful complex structures are discussed. These are exemplified for three complexes, and we show that the approach bridges the gap between theoretical docking approaches and complex NMR schemes for determining protein-ligand complexes; especially for relatively weak binders that do not lead to intermolecular NOEs.

Cyclic AMP-Dependent Protein Kinases↗
PubMed2004

NMR backbone assignment of a protein kinase catalytic domain by a combination of several approaches: application to the catalytic subunit of cAMP-dependent protein kinase.

Protein phosphorylation is one of the most important mechanisms used for intracellular regulation in eukaryotic cells. Currently, one of the best-characterized protein kinases is the catalytic subunit of cAMP-dependent protein kinase or protein kinase A (PKA). PKA has the typical bilobular structure of kinases, with the active site consisting of a cleft between the two structural lobes. For full kinase activity, the catalytic subunit has to be phosphorylated. The catalytic subunit of PKA has two main phosphorylation sites: Thr197 and Ser338. Binding of ATP or inhibitors to the ATP site induces large structural changes. Here we describe the partial backbone assignment of the PKA catalytic domain by NMR spectroscopy, which represents the first NMR assignment of any protein kinase catalytic domain. Backbone resonance assignment for the 42 kDa protein was accomplished by an approach employing 1) triply ((2)H,(13)C,(15)N) labeled protein and classical NMR assignment experiments, 2) back-calculation of chemical shifts from known X-ray structures, 3) use of paramagnetic adenosine derivatives as spin-labels, and 4) selective amino acid labeling. Interpretation of chemical-shift perturbations allowed mapping of the interaction surface with the protein kinase inhibitor H7. Furthermore, structural conformational changes were observed by comparison of backbone amide shifts obtained by 2D (1)H,(15)N TROSY of an inactive Thr197Ala mutant with the wild-type enzyme.

Amino Acid Sequence↗
PubMed2004

Novel techniques for weak alignment of proteins in solution using chemical tags coordinating lanthanide ions.

A molecule with an anisotropic magnetic susceptibility is spontaneously aligned in a static magnetic field. Alignment of such a molecule yields residual dipolar couplings and pseudocontact shifts. Lanthanide ions have recently been successfully used to provide an anisotropic magnetic susceptibility in target molecules either by replacing a calcium ion with a lanthanide ion in calcium-binding proteins or by attaching an EDTA derivative to a cysteine residue via a disulfide bond. Here we describe a novel enantiomerically pure EDTA derived tag that aligns stronger due to its shorter linker and does not suffer from stereochemical diversity upon lanthanide complexation. We observed residual (15)N,(1)H-dipolar couplings of up to 8 Hz at 800 MHz induced by a single alignment tensor from this tag.

Anisotropy↗