Martin Vogtherr

Design and Synthesis of Novel Lactate Dehydrogenase A Inhibitors by Fragment-Based Lead Generation

Lactate dehydrogenase A (LDHA) catalyzes the conversion of pyruvate to lactate, utilizing NADH as a cofactor. It has been identified as a potential therapeutic target in the area of cancer metabolism. In this manuscript we report our progress using fragment-based lead generation (FBLG), assisted by X-ray crystallography to develop small molecule LDHA inhibitors. Fragment hits were identified through NMR and SPR screening and optimized into lead compounds with nanomolar binding affinities via fragment linking. Also reported is their modification into cellular active compounds suitable for target validation work.

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 (2H,13C,15N) 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 1H,15N TROSY of an inactive Thr197Ala mutant with the wild‐type enzyme.

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.

NMR-based screening methods for lead discovery

Diversity and robustness of NMR based screening methods make these techniques highly attractive as tools for drug discovery. Although not all screening techniques discussed here may be applicable to any given target, there is however a good chance that at least one of the described methods will prove productive in finding several medium affinity ligands. A comparison of each of the methods is given in Table 1. For drug targets of molecular weight < 30 kDa SAR by NMR appears to be the method of choice since it yields detailed information about the location of the binding site. It remains to be seen whether 15N-1H-TROSY based screening techniques will prove useful for larger protein targets, especially considering the added effort needed for spectral assignment and the increased complexity due to spectral overlap. Nevertheless, with the application of new cryo-cooled NMR probes, 15N-1H-HSQC based screening can now be considered a high throughput method. Ligand-based NMR screening methods can be used for protein targets of virtually any size, but are restricted in the ligands binding affinity range. Because sufficient ligand-protein dissociation rates are needed, only binding of ligands with low (milimolar) to intermediate (micromolar) affinities is detectable. It is expected that cryo-cooled NMR probe technology will also advance ligand detected NMR screening to the high throughput level. Certainly protein and ligand concentrations can be lowered drastically and experiment times can be shortened with increased sensitivity. However, spectral overlap will be of major concern when mixtures of up to 100 compounds are to be screened. For such applications only techniques for which the signals of bound ligands survive will be useful, and sophisticated software will be needed to deconvolute the spectra of multiple bound ligands. Although only ligands with medium to low affinities can be found, ligand based NMR screening has been used as an effective prescreening tool for assay based high throughput screening. Identifying a large ensemble of medium affinity ligands may not only aid in building a binding site pharmacophore model (see Chapter 11), but also may yield crucial information for overcoming tissue availability, toxicity, or even intellectual property related problems. Although NMR based screening is only one of the more recent additions to the bag of tools used in drug discovery [1, 2], its simplicity and wide range of application (including protein-protein and protein-nucleic acid interactions) has attracted much attention. Advances in NMR instrumentation and methodology have already paved the road for NMR based screening to become a high throughput technique. In addition to this, NMR is exceptional in the amount of detailed structural [table: see text] information it can provide. Not only can NMR readily reveal the binding site (15N-1H-HSQC screening) or the conformation of the bound ligand (transfer NOE), but it can also supply information that enables precise docking of the ligand to the proteins binding pocket (isotope-filtered NOESY). NMR data can therefore provide a natural connection between experimental HTS and combinatorial chemistry techniques with computational methods such as 3D-database searching (see Chapter 10), virtual screening (docking) and structure-based ligand design (see also Chapter 8).

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.

Letter to the Editor: Assignment of the 1H, 13C and 15N resonances of the PPIase domain of the trigger factor from Mycoplasma genitalium

Tatjana N. Paraca,b, Martin Vogtherrc, Marcus Maurerd, Andreas Pahle, Heinz Ruterjansf, Christian Griesingera,g & Klaus Fiebigc,∗ aInstitut fur Organische Chemie der Universitat Frankfurt, Marie-Curie-Str. 11, 60439 Frankfurt, Germany; bK.U. Leuven, Department of Chemistry, Celestijnenlaan 200F, B-3001 Heverlee, Belgium; cMRPharm, MarieCurie-Str. 11, D-60439 Frankfurt, Germany; dAstaMedica, Weismullerstr. 45, D-60314 Frankfurt, Germany; eInstitut fur Pharmakologie und Toxikologie, Universitat Erlangen, Germany; fInstitut fur Biophysikalische Chemie der Universitat Frankfurt, Marie-Curie-Str. 11, D-60439 Frankfurt, Germany; gMax-Planck-Institut fur Biophysikalische Chemie, Am Fasberg 11, D-37077 Gottingen, Germany