Key aspects of the Novartis compound collection enhancement project for the compilation of a comprehensive chemogenomics drug discovery screening collection.

The NIBR (Novartis Institutes for BioMedical Research) compound collection enrichment and enhancement project integrates corporate internal combinatorial compound synthesis and external compound acquisition activities in order to build up a comprehensive screening collection for a modern drug discovery organization. The main purpose of the screening collection is to supply the Novartis drug discovery pipeline with hit-to-lead compounds for today's and the future's portfolio of drug discovery programs, and to provide tool compounds for the chemogenomics investigation of novel biological pathways and circuits. As such, it integrates designed focused and diversity-based compound sets from the synthetic and natural paradigms able to cope with druggable and currently deemed undruggable targets and molecular interaction modes. Herein, we will summarize together with new trends published in the literature, scientific challenges faced and key approaches taken at NIBR to match the chemical and biological spaces.

Binding-linked protonation of a DNA minor-groove agent.

The energetics for binding of a diphenyl diamidine antitrypanosomal agent CGP 40215A to DNA have been studied by spectroscopy, isothermal titration calorimetry, and surface plasmon resonance biosensor methods. Both amidines are positively charged under experimental conditions, but the linking group for the two phenyl amidines has a pK(a) of 6.3 that is susceptible to a protonation process. Spectroscopic studies indicate an increase of 2.7 pK(a) units in the linking group when the compound binds to an A/T minor-groove site. Calorimetric titrations in different buffers and pH conditions support the proton-linkage process and are in a good agreement with spectroscopic titrations. The two methods established a proton-uptake profile as a function of pH. The exothermic enthalpy of complex formation varies with different pH conditions. The observed binding enthalpy increases as a function of temperature indicating a negative heat capacity change that is typical for DNA minor-groove binders. Solvent accessible surface area calculations suggest that surface burial accounts for about one-half of the observed intrinsic negative heat capacity change. Biosensor and calorimetric experiments indicate that the binding affinities vary with pH values and salt concentrations due to protonation and electrostatic interactions. The surface plasmon resonance binding studies indicate that the charge density per phosphate in DNA hairpins is smaller than that in polymers. Energetic contributions from different factors were also estimated for the ligand/DNA complex.

New cationic lipids form channel-like pores in phospholipid bilayers.

Two representatives of a new class of cationic lipids were found to have high pore-forming activity in planar bilayer membranes. These molecules, called BHHD-TADC and BHTD-TADC, have qualitatively similar effects on phospholipid membranes. Addition of 2.5-5 micro M of either of them to the membrane bathing solutions resulted in formation of long-lived anion-selective pores with conductance in the range 0.1-2 nS in 0.1 M KCl. Pore formation was found to be dependent on the potential applied to the membrane. When negative potential was applied to membrane at the side of addition, the rate of pore formation was much lower compared to when the positive potential was applied. Dependence of pore formation on compound concentration was highly nonlinear, indicating that this process requires assembly of molecules in the membrane. Addition of any of these compounds on both sides of the membrane increased the efficiency of pore formation by one to two orders of magnitude. Pore formation was strongly pH dependent. Although pores were formed with high efficiency at pH 6.5, only occasional fluctuations of membrane conductance were observed at pH 7.5. Possible mechanisms of new compounds biological activity are discussed.

Characterization of a novel DNA minor-groove complex.

Many dicationic amidine compounds bind in the DNA minor groove and have excellent biological activity against a range of infectious diseases. Para-substituted aromatic diamidines such as furamidine, which is currently being tested against trypanosomiasis in humans, and berenil, which is used in animals, are typical examples of this class. Recently, a meta-substituted diamidine, CGP 40215A, has been found to have excellent antitrypanosomal activity. The compound has a linear, conjugated linking group that can be protonated under physiological conditions when the compound interacts with DNA. Structural and molecular dynamics analysis of the DNA complex indicated an unusual AT-specific complex that involved water-mediated H-bonds between one amidine of the compound and DNA bases at the floor of the minor groove. To investigate this unique system in more detail DNase I footprinting, surface plasmon resonance biosensor techniques, linear dichroism, circular dichroism, ultraviolet-visible spectroscopy, and additional molecular dynamics simulations have been conducted. Spectrophotometric titrations of CGP 40215A binding to poly(dAT)(2) have characteristics of DNA-binding-induced spectral changes as well as effects due to binding-induced protonation of the compound linker. Both footprinting and surface plasmon resonance results show that this compound has a high affinity for AT-rich sequences of DNA but very weak binding to GC sequences. The dissociation kinetics of the CGP 40215A-DNA complex are much slower than with similar diamidines such as berenil. The linear dichroism results support a minor-groove complex for the compound in AT DNA sequences. Molecular dynamics studies complement the structural analysis and provide a clear picture of the importance of water in mediating the dynamic interactions between the ligand and the DNA bases in the minor groove.