Identification of structural-kinetic and structural-thermodynamic relationships for thrombin inhibitors.

To improve our understanding of drug-target interactions, we explored the effect of introducing substituted amine residues with increased chain length in the P3 residue of the thrombin inhibitor melagatran. Inhibition, kinetic, and thermodynamic data obtained via stopped-flow spectroscopy (SF), isothermal microcalorimetry (ITC), and surface plasmon resonance (SPR) biosensor analysis were interpreted with the help of X-ray crystal structures of the enzyme-inhibitor complexes. The association rate became faster when the lipophilicity of the inhibitors was increased. This was coupled to an increased enthalpic component and a corresponding decreased entropic component. The dissociation rates were reduced with an increase in chain length, with only a smaller increase and a decrease in the enthalpic and entropic components, respectively. Overall, the affinity increased with an increase in chain length, with similar changes in the enthalpic and entropic components. ITC analysis confirmed the equilibrium data from SPR analysis, showing that the interaction of melagatran was the most enthalpy-driven interaction. Structural analysis of the thrombin-inhibitor complex showed that the orientation of the P1 and P2 parts of the molecules was very similar, but that there were significant differences in the interaction between the terminal part of the P3 side chain and the binding pocket. A combination of charge repulsion, H-bonds, and hydrophobic interactions could be used to explain the observed kinetic and thermodynamic profiles for the ligands. In conclusion, changes in the structure of a lead compound can have significant effects on its interaction with the target that translate directly into kinetic and thermodynamic effects. In contrast to what may be intuitively expected, hydrogen bond formation and breakage are not necessarily reflected in enthalpy gains and losses, respectively.

Active Site Mapping of an Aspartic Protease by Multiple Fragment Crystal Structures: Versatile Warheads To Address a Catalytic Dyad.

Crystallography is frequently used as follow-up method to validate hits identified by biophysical screening cascades. The capacity of crystallography to directly screen fragment libraries is often underestimated, due to its supposed low-throughput and need for high-quality crystals. We applied crystallographic fragment screening to map the protein-binding site of the aspartic protease endothiapepsin by individual soaking experiments. Here, we report on 41 fragments binding to the catalytic dyad and adjacent specificity pockets. The analysis identifies already known warheads but also reveals hydrazide, pyrazole, or carboxylic acid fragments as novel functional groups binding to the dyad. A remarkable swapping of the S1 and S1' pocket between structurally related fragments is explained by either steric demand, required displacement of a well-bound water molecule, or changes of trigonal-planar to tetrahedral geometry of an oxygen functional group in a side chain. Some warheads simultaneously occupying both S1 and S1' are promising starting points for fragment-growing strategies.

The versatile nature of the 6-aminoquinolone scaffold: identification of submicromolar hepatitis C virus NS5B inhibitors.

We have previously reported that the 6-aminoquinolone chemotype is a privileged scaffold to obtain antibacterial and antiviral agents. Herein we describe the design, synthesis, and enzymatic and cellular characterization of new 6-aminoquinolone derivatives as potent inhibitors of NS5B polymerase, an attractive and viable therapeutic target to develop safe anti-HCV agents. The 6-amino-7-[4-(2-pyridinyl)-1-piperazinyl]quinolone derivative 8 proved to be the best compound of this series, exhibiting an IC50 value of 0.069 µM against NS5B polymerase and selective antiviral effect (EC50 = 3.03 µM) coupled with the absence of any cytostatic effect (CC50 > 163 µM; SI > 54) in Huh 9-13 cells carrying a HCV genotype 1b, as measured by MTS assay. These results indicate that the 6-aminoquinolone scaffold is worthy of further investigation in the context of NS5B-targeted HCV drug discovery programs.

Resolution of the interaction mechanisms and characteristics of non-nucleoside inhibitors of hepatitis C virus polymerase.

Development of allosteric inhibitors into efficient drugs is hampered by their indirect mode-of-action and complex structure-kinetic relationships. To enable the design of efficient allosteric drugs targeting the polymerase of hepatitis C virus (NS5B), the interaction characteristics of three non-nucleoside compounds (filibuvir, VX-222, and tegobuvir) inhibiting HCV replication via NS5B have been analyzed. Since there was no logical correlation between the anti-HCV replicative and enzyme inhibitory effects of the compounds, surface plasmon resonance biosensor technology was used to resolve the mechanistic, kinetic, thermodynamic and chemodynamic features of their interactions with their target and their effect on its interaction with RNA. Tegobuvir could not be seen to interact with NS5B at all while filibuvir interacted in a single reversible step (except at low temperatures) and VX-222 in two serial steps, interpreted as an induced fit mechanism. Both filibuvir and VX-222 interfered with the interaction between NS5B and RNA. They competed for binding to the enzyme, suggesting that they had a common inhibition mechanism and identical or overlapping binding sites. The greater anti-HCV replicative activity of VX-222 over filibuvir is hypothesized to be due to a greater allosteric conformational effect, resulting in the formation of a less catalytically competent complex. In addition, the induced fit mechanism of VX-222 gives it a kinetic advantage over filibuvir, exhibited as a longer residence time. These insights have important consequences for the selection and optimization of new allosteric NS5B inhibitors.

Structure-based discovery of pyrazolobenzothiazine derivatives as inhibitors of hepatitis C virus replication.

The NS5B RNA-dependent RNA polymerase is an attractive target for the development of novel and selective inhibitors of hepatitis C virus replication. To identify novel structural hits as anti-HCV agents, we performed structure-based virtual screening of our in-house library followed by rational drug design, organic synthesis, and biological testing. These studies led to the identification of pyrazolobenzothiazine scaffold as a suitable template for obtaining novel anti-HCV agents targeting the NS5B polymerase. The best compound of this series was the meta-fluoro-N-1-phenyl pyrazolobenzothiazine derivative 4a, which exhibited an EC50 = 3.6 µM, EC90 = 25.6 µM, and CC50 > 180 µM in the Huh 9-13 replicon system, thus providing a good starting point for further hit evolution.

Experimental validation of a fragment library for lead discovery using SPR biosensor technology.

A new fragment library for lead discovery has been designed and experimentally validated for use in surface plasmon resonance (SPR) biosensor-based screening. The 930 compounds in the library were selected from 4.6 million commercially available compounds using a series of physicochemical and medicinal chemistry filters. They were screened against 3 prototypical drug targets: HIV-1 protease, thrombin and carbonic anhydrase, and a nontarget: human serum albumin. Compound solubility was not a problem under the conditions used for screening. The high sensitivity of the sensor surfaces allowed the detection of interactions for 35% to 97% of the fragments, depending on the target protein. None of the fragments was promiscuous (i.e., interacted with a stoichiometry ≥5:1 with all 4 proteins), and only 2 compounds dissociated slowly from all 4 proteins. The use of several targets proved valuable since several compounds would have been disqualified from the library on the grounds of promiscuity if fewer target proteins had been used. The experimental procedure allowed an efficient evaluation and exploration of the new fragment library and confirmed that the new library is suitable for SPR biosensor-based screening.

Quantification of normal cell fraction and copy number neutral LOH in clinical lung cancer samples using SNP array data.

BACKGROUND: Technologies based on DNA microarrays have the potential to provide detailed information on genomic aberrations in tumor cells. In practice a major obstacle for quantitative detection of aberrations is the heterogeneity of clinical tumor tissue. Since tumor tissue invariably contains genetically normal stromal cells, this may lead to a failure to detect aberrations in the tumor cells. PRINCIPAL FINDING: Using SNP array data from 44 non-small cell lung cancer samples we have developed a bioinformatic algorithm that accurately models the fractions of normal and tumor cells in clinical tumor samples. The proportion of normal cells in combination with SNP array data can be used to detect and quantify copy number neutral loss-of-heterozygosity (CNNLOH) in the tumor cells both in crude tumor tissue and in samples enriched for tumor cells by laser capture microdissection. CONCLUSION: Genome-wide quantitative analysis of CNNLOH using the CNNLOH Quantifier method can help to identify recurrent aberrations contributing to tumor development in clinical tumor samples. In addition, SNP-array based analysis of CNNLOH may become important for detection of aberrations that can be used for diagnostic and prognostic purposes.