Unified Software Solution for Efficient SPR Data Analysis in Drug Research.

Surface plasmon resonance (SPR) is a powerful method for obtaining detailed molecular interaction parameters. Modern instrumentation with its increased throughput has enabled routine screening by SPR in hit-to-lead and lead optimization programs, and SPR has become a mainstream drug discovery technology. However, the processing and reporting of SPR data in drug discovery are typically performed manually, which is both time-consuming and tedious. Here, we present the workflow concept, design and experiences with a software module relying on a single, browser-based software platform for the processing, analysis, and reporting of SPR data. The efficiency of this concept lies in the immediate availability of end results: data are processed and analyzed upon loading the raw data file, allowing the user to immediately quality control the results. Once completed, the user can automatically report those results to data repositories for corporate access and quickly generate printed reports or documents. The software module has resulted in a very efficient and effective workflow through saved time and improved quality control. We discuss these benefits and show how this process defines a new benchmark in the drug discovery industry for the handling, interpretation, visualization, and sharing of SPR data.

Fragment Screening of RORγt Using Cocktail Crystallography: Identification of Simultaneous Binding of Multiple Fragments.

The retinoic-acid-related orphan receptor γ t (RORγt), as a master regulator of Th17 cell pathology, has become an attractive target for small-molecule drug discovery for the treatment of Th17-cell-related autoimmune diseases. A crystallographic fragment screening was carried out for RORγt using the ligand binding domain. An overall hit rate of 5.5 % was obtained by screening 384 compounds in 96 cocktails. Five distinct hotspots were identified, and four regions of anchoring polar interactions were observed. In addition, significant induced fit was found for the binding of several fragments. Strikingly, a simultaneous binding of three fragments was revealed which presents interesting features including π-π stacking, multiple hydrogen bonds to the protein, and significant induced fit. Overall, the results offer a complete mapping of the ligand binding pocket and provide valuable inspiration in structure-based design for RORγt lead generation and optimization. The crystallographic screening also resulted in fragment hits that bind at the surface away from the ligand binding pocket. This surface site is near the plausible dimer interface by analogy with other nuclear receptor systems, which can provide initial hints to explore alternative ways to modulate RORγt through protein-protein interactions.

The proteasome deubiquitinase inhibitor VLX1570 shows selectivity for ubiquitin-specific protease-14 and induces apoptosis of multiple myeloma cells.

Inhibition of deubiquitinase (DUB) activity is a promising strategy for cancer therapy. VLX1570 is an inhibitor of proteasome DUB activity currently in clinical trials for relapsed multiple myeloma. Here we show that VLX1570 binds to and inhibits the activity of ubiquitin-specific protease-14 (USP14) in vitro, with comparatively weaker inhibitory activity towards UCHL5 (ubiquitin-C-terminal hydrolase-5). Exposure of multiple myeloma cells to VLX1570 resulted in thermostabilization of USP14 at therapeutically relevant concentrations. Transient knockdown of USP14 or UCHL5 expression by electroporation of siRNA reduced the viability of multiple myeloma cells. Treatment of multiple myeloma cells with VLX1570 induced the accumulation of proteasome-bound high molecular weight polyubiquitin conjugates and an apoptotic response. Sensitivity to VLX1570 was moderately affected by altered drug uptake, but was unaffected by overexpression of BCL2-family proteins or inhibitors of caspase activity. Finally, treatment with VLX1570 was found to lead to extended survival in xenograft models of multiple myeloma. Our findings demonstrate promising antiproliferative activity of VLX1570 in multiple myeloma, primarily associated with inhibition of USP14 activity.

The discovery of potent and selective non-steroidal glucocorticoid receptor modulators, suitable for inhalation.

We report the discovery of highly potent and selective non-steroidal glucocorticoid receptor modulators with PK properties suitable for inhalation. A high throughput screen of the AstraZeneca compound collection identified sulfonamide 3 as a potent non-steroidal glucocorticoid receptor ligand. Further optimization of this lead generated indazoles 30 and 48 that were progressed to characterization in in vivo models. X-ray crystallography was used to gain further insight into the binding mode of selected ligands.

X-ray structural analysis of tau-tubulin kinase†1 and its interactions with small molecular inhibitors.

Tau-tubulin kinase 1 (TTBK1) is a serine/threonine/tyrosine kinase that putatively phosphorylates residues including S422 in tau protein. Hyperphosphorylation of tau protein is the primary cause of tau pathology and neuronal death associated with Alzheimer's disease. A library of 12 truncation variants comprising the TTBK1 kinase domain was screened for expression in Escherichia coli and insect cells. One variant (residues 14-313) could be purified, but mass spectrometric analysis revealed extensive phosphorylation of the protein. Co-expression with lambda phosphatase in E. coli resulted in production of homogeneous, nonphosphorylated TTBK1. Binding of ATP and several compounds to TTBK1 was characterized by surface plasmon resonance. Crystal structures of TTBK1 in the unliganded form and in complex with ATP, and two high-affinity ATP-competitive inhibitors, 3-[(6,7-dimethoxyquinazolin-4-yl)amino]phenol (1) and methyl 2-bromo-5-(7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)benzoate (2), were elucidated. The structure revealed two clear basic patches near the ATP pocket providing an explanation of TTBK1 for phosphorylation-primed substrates. Interestingly, compound 2 displayed slow binding kinetics to TTBK1, the structure of TTBK1 in complex with this compound revealed a reorganization of the L199-D200 peptide backbone conformation together with altered hydrogen bonding with compound 2. These conformational changes necessary for the binding of compound 2 are likely the basis of the slow kinetics. This first TTBK1 structure can assist the discovery of novel inhibitors for the treatment of Alzheimer's disease.

The use of TrkA-PathHunter assay in high-throughput screening to identify compounds that affect nerve growth factor signaling.

The TrkA-PathHunter cell-based assay was used in high-throughput screening (HTS) to identify compounds that inhibit nerve growth factor (NGF)/TrkA signaling. The assay was conducted in a 384-well format, and typical Z' values during HTS ranged from 0.3 to 0.8. The reproducibility of IC50 values was good, and the use of cryopreserved cells was well tolerated, as judged by assay parameters such as Z' and S/B and by comparison of IC50 values obtained with cells in culture. During hit deconvolution, TrkA-kinase inhibitors were identified with ATP-competitive as well as non-ATP-competitive mechanisms of action. Furthermore, other mechanisms of action such as NGF and TrkA antagonists were also identified. Because of the different molecular mechanisms identified, it is possible that subsequent optimization work to increase affinity and selectivity might lead to compounds that could have a better chance to evoke clinical efficacy without the adverse effects observed for nonselective TrkA inhibitors.

Thermodynamic studies of ligand binding to the human homopentameric glycine receptor using isothermal titration calorimetry.

In this work, we describe a process for production of a Pichia pastoris strain which overproduces large quantities of the human glycine receptor. Subsequent purification yielded functional, uniform protein with expression yields of up to 5 mg per liter cell culture. As the wild-type protein is prone to proteolytic degradation, the labile sites were removed by mutagenesis resulting in an intracellular loop 2 deletion mutant with N-terminal modifications. This variant of the receptor is both stable during purification and storage on ice for up to a week as a complex with an antagonist. The quality of the protein is suitable for biophysical characterization and structural studies. The interaction of the agonist glycine and the antagonist strychnine with purified protein was analyzed by isothermal titration calorimetry. Strychnine binding is driven enthalpically with a K(D) of 138 ± 55 nM, a ΔH of -9708 ± 1195 cal/mol and a ΔS of -1.0 ± 4.1 cal/mol/K, whereas glycine binding is driven by entropy with a K(D) of 3.2 ± 0.8 µM, a ΔH of -2228 ± 1012 cal/mol and ΔS of 17.7 ± 2.8 cal/mol/K. Strychnine and glycine binding is competitive with a stoichiometry of one ligand molecule to one pentameric glycine receptor.

Structure-based design of 7-azaindole-pyrrolidine amides as inhibitors of 11ß-hydroxysteroid dehydrogenase type I.

Indole-pyrrolidines were identified as inhibitors of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) by high-throughput screening. Optimisation of the initial hit through structure-based design led to 7-azaindole-derivatives, with the best analogues displaying single digit nanomolar IC(50) potency. The modeling hypotheses were confirmed by solving the X-ray co-crystal structure of one of the lead compounds. These compounds were selective against 11ß-hydroxysteroid dehydrogenase type 2 (selectivity ratio >200) and exhibited good inhibition of 11ß-HSD1 (IC(50)<1µM) in a cellular model (3T3L1 adipocytes).

Monitoring of lipid storage in Caenorhabditis elegans using coherent anti-Stokes Raman scattering (CARS) microscopy.

Better understanding of the fundamental mechanisms behind metabolic diseases requires methods to monitor lipid stores on single-cell level in vivo. We have used Caenorhabditis elegans as a model organism to demonstrate the limitations of fluorescence microscopy for imaging of lipids compared with coherent anti-Stokes Raman scattering (CARS) microscopy, the latter allowing chemically specific and label-free imaging in living organisms. CARS microscopy was used to quantitatively monitor the impact of genetic variations in metabolic pathways on lipid storage in 60 specimens of C. elegans. We found that the feeding-defective mutant pha-3 contained a lipid volume fraction one-third of that found in control worms. In contrast, mutants (daf-2, daf-4 dauer) with deficiencies in the insulin and transforming growth factors (IGF and TGF-beta) signaling pathways had lipid volume fractions that were 1.4 and 2 times larger than controls, respectively. This was observed as an accumulation of small-sized lipid droplets in the hypodermal cells, hosting as much as 40% of the total lipid volume in contrast to the 9% for the wild-type larvae. Spectral CARS microscopy measurements indicated that this is accompanied by a shift in the ordering of the lipids from gel to liquid phase. We conclude that the degree of hypodermal lipid storage and the lipid phase can be used as a marker of lipid metabolism shift. This study shows that CARS microscopy has the potential to become a sensitive and important tool for studies of lipid storage mechanisms, improving our understanding of phenomena underlying metabolic disorders.