Tipos de indicadores

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6. Materials and Methods

6.1 Biological analytical methods

6.1.1 Molecular modelling

The co-crystal structure of SLF, FKBP51 or Compound 3a with the FK1 domain of FKBP51 were obtained from Dr. Andreas Bracher in Prof. Ulich Hartl `s group at Max Planck Institute of Biochemistry. Two of these structures were later published (4DRK and 3O5R)134, 209_.

All computer simulations were performed on Dell computer AMD athlon™64x2 dual core processor 3800+ 2.00GHz, 960MB RAM. Microsoft windows XP professional version 2002 service pack 2.

6.1.2 Molecular modelling of FKBP51 with bicyclic derivatives 7, 8, 42

The bicyclic [3.3.1] aza-amide nucleus 7, the bicyclic [4.3.1] aza-amide nucleus 8 and the C8_{-derivative bicyclic [4.3.1] aza-amide derivatives}

42 were constructed with Chemdraw 3D ultra 10.1. The structures were first drawn and cleaned up followed by energy calculation and minimization by MM2 computations with the minimum RMS gradient value at 0.1. Then, the C1-C6, N7, O1 and O10 of the bicyclic [3.3.1] aza- amide nucleus or the C1-C6, N7, O1 and O11 of the bicyclic [4.3.1] aza-amide nucleus was aligned and overlaid with corresponding atoms of 2 in the cocrystal structure of 2 and FKBP51. The resulting structures were saved as pdb files and visualized in PyMol.

6.1.3 Competition Binding Fluorescence Polarization Assay

The competition binding fluorescence polarization assay was performed as described141 _{under the guidance of Dr. Christian Kozany and Bastiaan Hoogeland.} Fluorescence polarization (FP) assays are widely used in high throughput screening in drug discovery. The flurophore-labeled ligand with size less than 5000 Da210 is

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excited by polarized light and emit depolarized light due to the rapid molecular motion of the flurophore during its fluorescence lifetime. This is usually in the nanosecond range and defined as the period between absorption of an excitation photon and the emission of a photon through fluorescence (Figure 14). If the flurophore-labeled ligand binds to a receptor of significantly greater size, the rotation of flurophore compared to the fluorescence lifetime is severely slowed down which causes less depolarization of the original plane of polarization. The extent of binding can be quantified by measuring the extent of depolarization.

Figure 14: (a) and (b) Scheme of FP assay mechanism. (c) Scheme of a competition binding FP assays.

In competition binding FP assays, an inhibitor competes with flurophore-labeled ligand in binding for a receptor which results in the increase of free flurophore-labeled ligand in solution. Thereby relatively less polarized light is emitted. Titration of the flurophore-labeled ligand and receptor complex with the inhibitor gives the relative binding affinity (IC50) of the inhibitor. The competition binding FP assay allows the determination of binding affinity of inhibitors from low nanomolar to high micromole range quickly and reproducibly.

+ ₊

Competitor

Fluo-ligand+receptor Competitor+receptor Fluo-ligand

High polarization Low polarization

Polarized exitation light Emitted light is depolarized Polarized exitation light Emitted light remains polarized Rapid rotation slow rotation Fluo-ligand Fluo-ligand+receptor a: b: c:

6. Materials and Methods

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6.1.4 Isothermal Titration Calorimetry experiments

The Isothermal Titration Calorimetry experiments were performed by Anne-Katrin Fabian.

Bacterially expressed, affinity purified human HisFKBP51FK1 (aa 1-140)141 was dialysed against ITC buffer (20mM HEPES pH=8, 150mM NaCl, 5% DMSO). The activity was confirmed by active site titration in an FP Assay as described before141_. The pH of protein was determined and ligand solutions were degassed and matched within 0.02 pH units.

ITC experiments were performed with a MicroCal iTC200 titration microcalorimeter (GE Healthcare). All experiments were conducted at 20°C. Compound 3d (1mM) was measured by injection into the measurement cell containing the protein (89µM). Due

to the limiting solubility compounds 2d and 4d were measured in a reverse setup injecting the protein (0.5mM and 0.16mM, respectively) into a solution of the ligand (40µM for 2d, 15µM for 4d). Heats of dilution were measured in blank titrations and subtracted from the binding heat values. ORIGIN software (version 7.0 Microcal) was used for data collection and analysis.

6.1.5 GR hormone binding assay.

The GR hormone binding assay was performed by Alexander Kirschner.

6.1.6 Crystallography

The crystallography was performed by Dr. Andreas Bracher as described209.

6.1.7 Reference compounds 5, 6b , 6c and 6h

The polycyclic compounds 5 and monocyclic compounds 6b , 6c and 6h were prepared by Christoph Kress and Ranganath Gopalakrishnan.

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7. Experimental Section

7.1 General chemical methods

All reactions were preformed in flame-dried glassware fitted with rubber septa under argon unless otherwise noted. Air- and moisture-sensitive liquids were transferred via syringe. Organic solvents were dried over MgSO4 and concentrated by rotary evaporation.

7.1.1 Nuclear Magnetic Resonance (NMR)

The NMR measurements were performed by Claudia Dubler and Dr. David S. Stephenson.

The 1_H, 13_{C-NMR-spectra, 2D HSQC, HMBC, COSY and NOESY were recorded on} a Bruker AC 300, Bruker XL 400 or Bruker AMX 600 at room temperature at the NMR-facility, Department of Chemistry and Pharmacy, Ludwig-Maximilians- Universitaet Muenchen. Chemical shifts were reported in δ values (ppm); the

hydrogenated residues of deuterated solvent were used as internal standard (CDCl3:

δ = 7.26 ppm in 1H NMR and δ = 77 ppm in 13C NMR). Signals were described as s,

d, t, and m for singlet, doublet, triplet and multiplet respectively. All coupling constants (J) were given in Hz.

7.1.2 Mass Spectrometry

The Mass spectra (m/z) were obtained on a Thermo Finnigan LCQ DECA XP Plus mass spectrometer (ESI) at the Max Planck Institute of Psychiatry while the high resolution mass spectrometry was carried out by Elisabeth Weyher at MPI for Biochemistry (Microchemistry Core facility) on Varian Mat711 mass spectrometer (ESI) or on a JMS GCmate II JEOL mass spectrometer (EI) by Dr. Lars Allmendinger at the Department of Chemistry and Pharmacy, Ludwig-Maximilians-University Munich.