Exponentially growing MT-4 cells were seeded at an initial selleck kinase inhibitor density of 1 × 105 cells/ml in 96-well plates in RPMI-1640 medium, supplemented with 10 % fetal bovine serum (FBS), 100 units/ml penicillin G, and 100 μg/ml streptomycin. Cell cultures were then incubated at 37 °C in a humidified 5 % CO2 atmosphere in the absence or presence

of serial dilutions of test compounds. Cell viability was determined after 96 h at 37 °C by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) method (Pauwels et al., 1988). Antiviral assays Compound’s activity against HIV-1 was based on inhibition of virus-induced cytopathogenicity in MT-4 cell acutely infected with a multiplicity of infection (m.o.i.) of 0.01. In brief, 50 μl of RPMI containing 1 × 104 MT-4 cells were added to each well of flat-bottom microtitre trays, containing 50 μl of RPMI with or without serial dilutions of test compounds. Then, 20 μl of a HIV-1 suspension containing 100 CCID50

was added. After a 4-day incubation at 37 °C, cell viability was determined by the MTT method (Pauwels et al., 1988). In vitro ligand binding assays Ligand Seliciclib purchase studies with native 5-HT1A receptor were conducted according to the methods previously described (RG-7388 price Lewgowd et al., 2011). X-ray structure determination Suitable crystals were mounted for measurements. The X-ray measurements were performed at 100(2) K on a KUMA CCD k-axis diffractometer with graphite-monochromated Mo Kα radiation (0.71073 Å). The crystals were positioned at 62.25 mm from the KM4CCD camera. The data were corrected for Lorentz and polarization effects, additionally absorption corrections were applied. Data reduction and analysis were carried out with the Kuma Diffraction (Wrocław, Poland) programmes (Oxford Diffraction CrysAlis CCD and CrysAlis RED, 2001). The structures were solved by direct methods (Sheldrick, 1990) and refined by using

SHELXL (Sheldrick, 1997) The refinement was based on F 2 for all reflections except for those with very negative F 2. The weighted R factor, wR, and all goodness-of-fit S values are based on F 2. The non-hydrogen atoms were refined anisotropically. The hydrogen atoms were located from a difference map and were refined isotropically. The atomic scattering factors were taken from the International Tables (Wilson, 1992). Immune system Crystallographic data for the structures have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC 913714-913719. Copy of the data can be obtained on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (email: [email protected]). X-ray crystal data for 2 C37H28BrNO3, monoclinic space group P21/c: a = 15.7066(8), b = 7.9750(4), c = 23.0807(12) Å, β = 100.366(4); V = 2843.9(3) Å3, Z = 4, D calcd = 1.435 g/cm3; μ = 1.485 mm−1; F(000) = 1264. A total of 21,137 reflections were integrated in the θ-range of 2.71°–25.0° of which 5,007 were unique, leaving an overall R-merge of 0.041.