Discovery of 5-naphthylidene-2,4-thiazolidinedione derivatives as selective HDAC8 inhibitors and evaluation of their cytotoxic effects in leukemic cell lines.

Histone deacetylases (HDACs) are being explored as a therapeutic target for interventions in different types of cancer. HDAC8 is a class I HDAC that is implicated as a therapeutic target in various indication areas, including different types of cancer and particularly childhood neuroblastoma. Most previously described HDAC8-selective inhibitors contain a hydroxamate function as zinc binding group (ZBG) to confer potency. However, hydroxamate class HDAC inhibitors have raised increasing concerns about their mutagenic character. Therefore, non-hydroxamate based inhibitors could prove to be safer than hydroxamates. In the present work, a series of novel 5-naphthylidene-2,4-thiazolidinedione was designed and evaluated as potential antiproliferative agents targeting selectively HDAC8 enzyme. Eleven novel derivatives were synthesized, purified and characterized by spectroscopic techniques. Compounds 3k and 3h was found to be most potent selective inhibitors of HDAC8 with IC50 values of 2.7 µM and 6.3 µM respectively. 3a to 3i was found to be most cytotoxic in leukemic cell lines. 3a and 3 h both were found to induce apoptosis and cause cell cycle arrest in G2/M phase.

Nanobeads as a solid phase in a solute homogeneous assay format.

The measurement of numerous samples as in drug screening or diagnostics has been improved significantly over recent years. The processing of a great number of carriers with 96, 384 or 1536 wells is not the limiting step anymore and more than 100,000 samples can be analyzed within 24 h. New challenges arise in optimizing data quality and assay volume in order to gain more reliable results with a reduced consumption of assay reagents. Both are addressed with an approach using dispersed nanoparticles analyzed by a new detection technology, fluorescence intensity distribution analysis. This homogeneous assay is a generic format holding great potential in expanding the assay strategy portfolio for diagnostic and screening applications.

Enzyme inhibition assays using fluorescence correlation spectroscopy: a new algorithm for the derivation of kcat/KM and Ki values at substrate concentrations much lower than the Michaelis constant.

A new mathematical formalism is deduced which allows for the calculation of the k(cat) over K(M) ratio based on measurements of the enzyme kinetics with substrate concentrations much lower than K(M). The equations are also applied on the action of an inhibitor on enzyme activity yielding the binding constant, K(i), of an inhibitor molecule. For practical evaluation of the new theoretical approach, the enzymatic reaction of CD45 phosphatase was used as a well-characterized model system with known inhibitors for testing the K(i) value determination scheme. The k(cat)/K(M) ratio was calulated to be 4.7 x 10(5) M(-)(1) s(-)(1), the K(i) of the inhibitor molecule PKF52-524 was estimated to be (1-2) x 10(-)(7) M and the association rate of the inhibitor PKF52-524 to CD45 phosphatase was estimated to be 59 M(-)(1) s(-)(1).

A novel and robust homogeneous fluorescence-based assay using nanoparticles for pharmaceutical screening and diagnostics.

We have established a new type of homogeneous immunoassay based on nanoparticles (nanoparticle immunoassay, or NPIA) being analyzed using fluorescence intensity distribution analysis (FIDA). This method allows the characterization of single fluorescently labeled molecules or particles with respect to their molecular brightness and concentration. Upon binding of conjugates to molecules coupled to the nanoparticle surface, the brightness of the complex scales with the number of bound conjugates. The complexes can then be distinguished accurately from free conjugate and concentrations of free and bound molecules can be determined reliably. In this study we present various examples of NPIAs where capture antibodies were linked to the nanoparticles, which were either artificial beads or bacteria. Two assay formats have been developed; first, direct labeling of the conjugate was used to quantitate free antigen through competition experiments, and second, an antigen-directed antibody was labeled to establish an assay similar to a sandwich ELISA setup. The major advantages of a NPIA are the robustness and high signal-to-noise ratio at short measurement times, as demonstrated with a miniaturized experiment in a Nanocarriertrade mark holding a volume of 1 microl/well. In addition to the good data quality, NPIAs are straightforward to perform because they require no washing steps. NPIAs open new dimensions for high throughput pharmaceutical screening and diagnostics. Assay development times can be reduced significantly because of a simple toolbox principle that is applicable to most types of assays.

Mechanism of the alpha-complementation reaction of E. coli beta-galactosidase deduced from fluorescence correlation spectroscopy measurements.

The kinetics of the alpha-complementation reaction of two protein fragments yielding active E. coli beta-galactosidase was measured using fluorescence correlation spectroscopy (FCS). The association reaction was extremely slow with an apparent association rate kapp of 207 M-1 s-1. This low association rate can be explained by a fast pre-equilibrium and slow subsequent steps involving at least two dimeric complexes. The subsequent formation of a tetrameric complex is probable and consistent with the experimental data. The complexes comprise two or four subunits, respectively, of the large fragment (EA)2 and in all cases only one small fragment, ED which has been labeled with Cy5. These kinetics have been compared to the association kinetics of ED to inactivated (EA)2. The kinetics were similar to the association with native (EA)2. The data support the observation that lyophilization of (EA)2 in a reducing environment which causes complete loss of enzymatic activity does not interfere with binding.

Fluorescence cross-correlation: a new concept for polymerase chain reaction.

In this article we present a new concept for the detection of any specifically amplified target DNA sequences in multiple polymerase chain reactions (PCR) based on fluorescence correlation spectroscopy (FCS). The accumulation of double-stranded target DNA is monitored by the cross-correlated fluorescence signals provided by two amplification primers which are 5'-tagged with two different kinds of fluorophores (Rhodamine-Green and Cy5). Only the amplified target DNA sequence carrying both primers is observed. Its signal emerges from the background of non-incorporated or non-specifically incorporated primers. Down to 10-25 initial copy numbers of the template in the PCR compartment DNA can presently be detected. No external or internal standards are required for determining the size and the amplified copy number of specific DNA. The PCR amplification process is started with all ingredients in a single compartment (e.g. of a microtiter plate), in which amplification and measurement are performed. This eliminates the need for post-PCR purification steps. The homogeneous one-tube approach does not depend on fluorescence energy transfer between the fluorogenic dyes. Thus, it does not interfere with the enzymatic amplification reaction of PCR and allows the continued use of different conditions for amplifying DNA. The results exemplified by PCR-amplified 217-bp and 389-bp target DNA sequences demonstrate that the analysis based on two-color fluorescence cross-correlation is a powerful method for simplifying the identification of targets in PCR for medical use. For this purpose, an instrument optimized for two-color excitation and detection of two-color emission has been developed, incorporating the principle of confocal arrangement.

The cyclic AMP receptor promoter DNA complex: a comparison of crystal and solution structure by quantitative molecular electrooptics.

The complexes formed between the cyclic AMP receptor and three different promoter DNA fragments, including a synthetic 30 bp fragment with the sequence used for determination of the crystal structure, have been analysed in solution by measurements of the electric dichroism (ED) at an ionic strength of 105 mM, using a special instrument based on cable discharge. The ED of the protein is negligible and, thus, the ED of the complexes is determined by the DNA and its orientation relative to the protein. The complex formed between the cyclic AMP receptor with the 30 bp fragment is characterized by a positive ED, indicating that the electric dipole is perpendicular relative to the direction of the helix; moreover, the dipole changes its nature from an induced one for the free DNA to a permanent one of 3.0 x 10(-27) Cm for the complex; both the limiting value of the ED +0.3 and the dichroism decay time constant of 62 ns found for the complex (free DNA: 52 ns; 20 degrees C) demonstrate bending of the DNA double helix. All these parameters are calculated quantitatively from the crystal structure: bead model simulations are used to derive the coefficients of rotational diffusion and to define the center of diffusion, which is the reference for calculation of the dipole vector; the dipole vector is then the basis for calculation of the limit value of the dichroism; the time constants are derived from the diffusion coefficients of the bead model The calculated parameters are in very satisfactory agreement with the experimental ones, demonstrating agreement of the structures in the crystal and in solution with respect to their essential features. These results also demonstrate the utility of electrooptical procedures for a quantitative comparison of crystal or model structures with structures in solution. While the crystal structure has been determined only for the complex with the 30 bp promoter fragment, it has been relatively simple to extend measurements of the ED to complexes formed with a 40 bp and a 203 bp promoter fragment: the data obtained for a 40 bp fragment with the consensus binding sequence are quite similar to those obtained for the 30 bp promoter, whereas the data obtained for the 203 bp promoter clearly show a much higher degree of protein induced bending with a bending angle of approximately 180 degrees.

Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution.

The present paper describes a new experimental scheme for following diffusion and chemical reaction systems of fluorescently labeled molecules in the nanomolar concentration range by fluorescence correlation analysis. In the dual-color fluorescence cross-correlation spectroscopy provided here, the concentration and diffusion characteristics of two fluorescent species in solution as well as their reaction product can be followed in parallel. By using two differently labeled reaction partners, the selectivity to investigate the temporal evolution of reaction product is significantly increased compared to ordinary one-color fluorescence autocorrelation systems. Here we develop the theoretical and experimental basis for carrying out measurements in a confocal dual-beam fluorescence correlation spectroscopy setup and discuss conditions that are favorable for cross-correlation analysis. The measurement principle is explained for carrying out DNA-DNA renaturation kinetics with two differently labeled complementary strands. The concentration of the reaction product can be directly determined from the cross-correlation amplitude.

The structure of the RNA polymerase-promoter complex. DNA-bending-angle by quantitative electrooptics.

The complex formed between RNA polymerase holoenzyme from Escherichia coli and the strong promoter A1 from the phage T7 has been characterized by measurements of the electric dichroism. The dichroism decay time constant of a promoter DNA fragment with 126 bp increases upon binding of the polymerase, but the increase is less than expected for simple addition of the components at the known binding site. Our results demonstrate a protein-induced decrease of the hydrodynamic DNA dimensions, which is not a consistent with an increased flexibility, but indicates bending of the double helix with a relatively narrow distribution of bending angles. We have characterized the degree of DNA bending by bead model simulations and used, in addition to our present experimental data, the available information on the overall size and shape of the RNA polymerase, together with the location of the DNA bending center at the starting point of RNA synthesis. We conclude that the bending angle is 45 degrees (+/- 5 degrees).

Mechanism of intercalation into the DNA double helix by ethidium.

The mechanism of intercalation into DNA double helices by ethidium has been analyzed by temperature-jump relaxation and stopped-flow measurements using fluorescence detection. Artifacts due to field- or flow-induced alignment have been eliminated by measurements under magic angle conditions; the theoretical basis for suppression of orientation effects resulting from external forces is given for the case of fluorescence measurements. Excluded site effects have been avoided by restriction to low degrees of binding. The temperature-jump relaxation observed for ethidium binding to DNA could be described by single exponentials under most conditions. The reciprocal time constants increased linearly with the DNA concentration, leading to association rate constants of 2.7 x 10(6) M-1 s-1 at 12 degrees C. These rate constants are virtually independent of the DNA chain length for samples with 200, 500, 4228, and 30,000 base pairs, showing that the rate is controlled by reaction and not by a diffusive process. At high DNA concentrations around 200 microM, an additional relaxation effect with an amplitude opposite to the main one is observed which is probably due to some conformational change of the DNA-ethidium complex. The results obtained by stopped-flow measurements are consistent with those from T-jump measurements, but owing to higher amplitudes and better signal to noise ratios, the stopped-flow data clearly require two exponentials for satisfactory representation. The reciprocal time constants for both processes increase linearly with the DNA concentration. The simplest mechanism consistent with this result involves parallel formation of two different complexes with a direct transfer of ethidium between the binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)