Ralf Heilker

G-protein-coupled receptor-focused drug discovery using a target class platform approach.

In recent years, several large pharmaceutical companies have taken a novel approach to drug discovery biology and chemistry in that they channel their efforts with respect to particular target classes, such as G-protein-coupled receptors (GPCRs), toward dedicated, specialized teams. Benefits of such an organizational structure are the prospects of establishing several target-family-specific experimental techniques and skill sets, thereby enabling a comprehensive functional profiling of drug candidates in different pharmacological respects. In this context, the recently increased number of reports on GPCR ligand-biased signaling has further spurred the efforts in the pharmaceutical industry toward broader biological characterization of the test compounds, for example employing high-content screening to analyze different GPCR ligand-induced signaling pathways. The knowledge of the disease-relevant functional properties of the small molecule GPCR ligands enables target-specific chemical optimization and GPCR-subclass-directed library design. In the case of GPCRs, where little--although at present slowly expanding--structural information on the targets is available, the modeling of GPCR structures crucially depends on biological validation (typically supported by site-directed mutagenesis of the GPCR ligand binding site). In this review, we aim to recapitulate efforts in the pharmaceutical industry to address GPCR-directed drug discovery in a target-class-directed platform approach: establishing GPCR-specific biological assay panels and creating computational chemistry methods for finding and optimizing small molecules modulating the activity of GPCRs.

mRuby, a Bright Monomeric Red Fluorescent Protein for Labeling of Subcellular Structures

A monomeric variant of the red fluorescent protein eqFP611, mRuby, is described. With excitation and emission maxima at 558 nm and 605 nm, respectively, and a large Stokes shift of 47 nm, mRuby appears particularly useful for imaging applications. The protein shows an exceptional resistance to denaturation at pH extremes. Moreover, mRuby is about ten-fold brighter compared to EGFP when being targeted to the endoplasmic reticulum. The engineering process of eqFP611 revealed that the C-terminal tail of the protein acts as a natural peroxisomal targeting signal (PTS). Using an mRuby variant carrying the eqFP611-PTS, we discovered that ordered inheritance of peroxisomes is widespread during mitosis of different mammalian cell types. The ordered partitioning is realized by the formation of peroxisome clusters around the poles of the mitotic spindle and ensures that equal numbers of the organelle are inherited by the daughter cells. The unique spectral properties make mRuby the marker of choice for a multitude of cell biological applications. Moreover, the use of mRuby has allowed novel insights in the biology of organelles responsible for severe human diseases.

Photoconvertible Fluorescent Protein EosFP: Biophysical Properties and Cell Biology Applications

Abstract EosFP is a fluorescent protein from the coral Lobophyllia hemprichii that changes its fluorescence emission from green to red upon irradiation with near-UV light. Here we present the spectroscopic properties of wild-type EosFP and a variety of monomeric and dimeric mutants and provide a structural interpretation of its oligomerization and photoconversion, which is based on X-ray structure analysis of the green and red species that we reported recently. Because functional expression of the monomeric EosFP variant is limited to temperatures of 30°C, we have developed a tandem dimer. This construct, in which two EosFP subunits are connected by a flexible 12 amino acid linker, expresses well after fusion with the androgen and endothelin A receptors at 37°C. A variety of applications in cellular imaging, developmental biology and automated high-content screening applications are presented, which demonstrate that EosFP is a powerful tool for in vivo monitoring of cellular processes.

Confocal optics microscopy for biochemical and cellular high-throughput screening

In recent years, both academia and pharmaceutical industry have produced significant advances in confocal detection and spectroscopy by laser-induced fluorescence. Confocal fluorescence studies provide information on identity, size, diffusion coefficient and concentration of the fluorescently labeled entity. This enables the establishment of sophisticated biochemical drug screening assays using the multitude of fluorescence parameters that can be observed (e.g. molecular brightness, fluorescence lifetime, anisotropy, resonance energy transfer). In cellular screening assays, confocality introduces spatial resolution in the vertical direction and reduces background fluorescence from outside the focal plane. Confocal HTS systems focusing on femtoliter-sized observation volumes allow for assay volumes far beyond current limits.

Optimized and Far-Red-Emitting Variants of Fluorescent Protein eqFP611

Fluorescent proteins (FPs) emitting in the far-red region of the spectrum are highly advantageous for whole-body imaging applications because scattering and absorption of long-wavelength light is markedly reduced in tissue. We characterized variants of the red fluorescent protein eqFP611 with bright fluorescence emission shifted up to 639 nm. The additional red shift is caused by a trans-cis isomerization of the chromophore. The equilibrium between the trans and cis conformations is strongly influenced by amino acid residues 143 and 158. Pseudo monomeric tags were obtained by further genetic engineering. For the red chromophores of eqFP611 variants, molar extinction coefficients of up to approximately 150,000 were determined by an approach that is not affected by the presence of molecules with nonfunctional red chromophores. The bright fluorescence makes the red-shifted eqFP611 variants promising lead structures for the development of near-infrared fluorescent markers. The red fluorescent proteins performed well in cell biological applications, including two-photon imaging.

Safety and Efficacy of an Inhaled Epidermal Growth Factor Receptor Inhibitor (BIBW 2948 BS) in Chronic Obstructive Pulmonary Disease

RATIONALE Epidermal growth factor receptor (EGFR) activation is implicated in mucin hypersecretion in chronic obstructive pulmonary disease (COPD). OBJECTIVES To investigate the safety and efficacy of an inhaled EGFR antagonist (BIBW 2948) in COPD. METHODS Multicenter, double-blind, placebo-controlled trial of 4 weeks of treatment with two doses of BIBW 2948 (15 and 30 mg twice a day) on safety and mucin-related outcomes in 48 patients with COPD. The effect of BIBW 2948 on EGFR activation in airway epithelial cells was assessed using an ex vivo assay. Efficacy measures included the volume of mucin in the airway epithelium (Vs mu,bala) in bronchial biopsies and the expression of mucin genes in bronchial brushings. MEASUREMENTS AND MAIN RESULTS Inhaled BIBW 2948 induced a dose-related inhibition of EGFR internalization (reflecting decreased EGFR activation) in epithelial cells from treated subjects. However, BIBW 2948 was associated with a dose-related increase in adverse events, including reversible liver enzyme elevation (n = 2), and reduction in FEV(1). The changes in mucin stores and mucin gene expression were not significantly different in the pooled BIBW 2948 group versus placebo (volume of mucin per surface area of basal lamina = 0.22 +/- 7.11 vs. 0.47 +/- 8.06 microm(3)/microm(2); P = 0.93). However, in the 30 mg twice a day group, the reduction in epithelial mucin stores was greatest in subjects with the greatest degree of EGFR inhibition (Pearson r = 0.98; 95% confidence interval, 0.71-0.99). CONCLUSIONS Four-week treatment with BIBW 2948 did not significantly decrease epithelial mucin stores and was poorly tolerated in patients with COPD. Ex vivo analyses suggest that higher doses may be more effective at both EGFR inhibition and decreases in mucin stores but that adverse events should be expected. Clinical trial registered with www.clinicaltrials.gov (NCT00423137).

Trans-cis isomerization is responsible for the red-shifted fluorescence in variants of the red fluorescent protein eqFP611.

An important class of red fluorescent proteins (RFPs) feature a 2-iminomethyl-5-(4-hydroxybenzylidene)imidazolinone chromophore. Among these proteins, eqFP611 has the chromophore in a coplanar trans orientation, whereas the cis isomer is preferred by other RFPs such as DsRed and its variants. In the photoactivatable protein asFP595, the chromophore can even be switched from the nonfluorescent trans to the fluorescent cis state by light. By using X-ray crystallography, we have determined the structure of dimeric eqFP611 at high resolution (up to 1.1 A). In the far-red emitting eqFP611 variant d2RFP630, which carries an additional Asn143Ser mutation, the chromophore resides predominantly (approximately 80%) in the cis isomeric state, and in RFP639, which has Asn143Ser and Ser158Cys mutations, the chromophore is found completely in the cis form. The pronounced red shift of excitation and emission maxima of RFP639 can thus unambiguously be assigned to trans-cis isomerization of the chromophore. Among RFPs, eqFP611 is thus unique because its chromophore is highly fluorescent in both the cis and trans isomeric forms.

G protein-coupled receptor internalization assays in the high-content screening format.

High-content screening (HCS), a combination of fluorescence microscopic imaging and automated image analysis, has become a frequently applied tool to study test compound effects in cellular disease-modeling systems. This chapter describes the measurement of G protein-coupled receptor (GPCR) internalization in the HCS format using a high-throughput, confocal cellular imaging device. GPCRs are the most successful group of therapeutic targets on the pharmaceutical market. Accordingly, the search for compounds that interfere with GPCR function in a specific and selective way is a major focus of the pharmaceutical industry today. This chapter describes methods for the ligand-induced internalization of GPCRs labeled previously with either a fluorophore-conjugated ligand or an antibody directed against an N-terminal tag of the GPCR. Both labeling techniques produce robust assay formats. Complementary to other functional GPCR drug discovery assays, internalization assays enable a pharmacological analysis of test compounds. We conclude that GPCR internalization assays represent a valuable medium/high-throughput screening format to determine the cellular activity of GPCR ligands.

iPS cell derived neuronal cells for drug discovery.

Owing to the inherent disconnect between drug pharmacology in heterologous cellular models and drug efficacy in vivo, the quest for more predictive in vitro systems is one of the most urgent challenges of modern drug discovery. An improved pharmacological in vitro profiling would employ primary samples of the proper drug-targeted human tissue or the bona fide human disease-relevant cells. With the advent of induced pluripotent stem (iPS) cell technology the facilitated access to a variety of disease-relevant target cells is now held out in prospect. In this review, we focus on the use of human iPS cell derived neurons for high throughput pharmaceutical drug screening, employing detection technologies that are sufficiently sensitive to measure signaling in cells with physiological target protein expression levels.

An orally bioavailable positive allosteric modulator of the mGlu4 receptor with efficacy in an animal model of motor dysfunction

A high-throughput screening campaign identified 4-((E)-styryl)-pyrimidin-2-ylamine (11) as a positive allosteric modulator of the metabotropic glutamate (mGlu) receptor subtype 4. An evaluation of the structure-activity relationships (SAR) of 11 is described and the efficacy of this compound in a haloperidol-induced catalepsy rat model following oral administration is presented.