Probing Factor Xa Protein-Ligand Interactions: Accurate Free Energy Calculations and Experimental Validations of Two Series of High-Affinity Ligands.

The accurate prediction of protein-ligand binding affinity belongs to one of the central goals in computer-based drug design. Molecular dynamics (MD)-based free energy calculations have become increasingly popular in this respect due to their accuracy and solid theoretical basis. Here, we present a combined study which encompasses experimental and computational studies on two series of factor Xa ligands, which enclose a broad chemical space including large modifications of the central scaffold. Using this integrated approach, we identified several new ligands with different heterocyclic scaffolds different from the previously identified indole-2-carboxamides that show superior or similar affinity. Furthermore, the so far underexplored terminal alkyne moiety proved to be a suitable non-classical bioisosteric replacement for the higher halogen-π aryl interactions. With this challenging example, we demonstrated the ability of the MD-based non-equilibrium free energy calculation approach for guiding crucial modifications in the lead optimization process, such as scaffold replacement and single-site modifications at molecular interaction hot spots.

Small Molecule Inhibitor Targeting CDT1/Geminin Protein Complex Promotes DNA Damage and Cell Death in Cancer Cells.

DNA replication initiation requires the loading of MCM2-7 complexes at the origins of replication during G1. Replication licensing renders chromatin competent for DNA replication and its tight regulation is essential to prevent aberrant DNA replication and genomic instability. CDT1 is a critical factor of licensing and its activity is controlled by redundant mechanisms, including Geminin, a protein inhibitor of CDT1. Aberrant CDT1 and Geminin expression have been shown to promote tumorigenesis in vivo and are also evident in multiple human tumors. In this study, we developed an in vitro AlphaScreen™ high-throughput screening (HTS) assay for the identification of small-molecule inhibitors targeting the CDT1/Geminin protein complex. Biochemical characterization of the most potent compound, AF615, provided evidence of specific, dose-dependent inhibition of Geminin binding to CDT1 both in-vitro and in cells. Moreover, compound AF615 induces DNA damage, inhibits DNA synthesis and reduces viability selectively in cancer cell lines, and this effect is CDT1-dependent. Taken together, our data suggest that AF615 may serve as a useful compound to elucidate the role of CDT1/Geminin protein complex in replication licensing and origin firing as well as a scaffold for further medicinal chemistry optimisation.

Synthesis and Evaluation of Novel Ring-Strained Noncanonical Amino Acids for Residue-Specific Bioorthogonal Reactions in Living Cells.

Bioorthogonal reactions are ideally suited to selectively modify proteins in complex environments, even in vivo. Kinetics and product stability of these reactions are crucial parameters to evaluate their usefulness for specific applications. Strain promoted inverse electron demand Diels-Alder cycloadditions (SPIEDAC) between tetrazines and strained alkenes or alkynes are particularly popular, as they allow ultrafast labeling inside cells. In combination with genetic code expansion (GCE)-a method that allows to incorporate noncanonical amino acids (ncAAs) site-specifically into proteins in vivo. These reactions enable residue-specific fluorophore attachment to proteins in living mammalian cells. Several SPIEDAC capable ncAAs have been presented and studied under diverse conditions, revealing different instabilities ranging from educt decomposition to product loss due to ß-elimination. To identify which compounds yield the best labeling inside living mammalian cells has frequently been difficult. In this study we present a) the synthesis of four new SPIEDAC reactive ncAAs that cannot undergo ß-elimination and b) a fluorescence flow cytometry based FRET-assay to measure reaction kinetics inside living cells. Our results, which at first sight can be seen conflicting with some other studies, capture GCE-specific experimental conditions, such as long-term exposure of the ring-strained ncAA to living cells, that are not taken into account in other assays.

A time-resolved live cell imaging assay to identify small molecule inhibitors of FGF2 signaling.

Fibroblast growth factor 2 (FGF2) is a cell survival factor with crucial functions in tumor-induced angiogenesis. Here, we describe a novel time-resolved FGF2 signaling assay based upon live cell imaging of neuroblastoma cells. To validate this system, we tested 8960 small molecules for inhibition of FGF2 signaling with kinetic resolution. Hit compounds were validated in dose-response experiments for FGF2 signaling, FGF receptor antagonism, downstream ERK phosphorylation and FGF2-dependent chemoresistance in a cellular leukemia model system. The new screening system for FGF2 signaling inhibitors has unique features, deselecting compounds with pleiotropic effects on cell proliferation and, along with the experimental pipeline reported, great potential for the discovery of new classes of FGF2 signaling inhibitors that block FGF2 dependent tumor cell survival.

Small Molecule Inhibitors Targeting Tec Kinase Block Unconventional Secretion of Fibroblast Growth Factor 2.

Fibroblast growth factor 2 (FGF2) is a potent mitogen promoting both tumor cell survival and tumor-induced angiogenesis. It is secreted by an unconventional secretory mechanism that is based upon direct translocation across the plasma membrane. Key steps of this process are (i) phosphoinositide-dependent membrane recruitment, (ii) FGF2 oligomerization and membrane pore formation, and (iii) extracellular trapping mediated by membrane-proximal heparan sulfate proteoglycans. Efficient secretion of FGF2 is supported by Tec kinase that stimulates membrane pore formation based upon tyrosine phosphorylation of FGF2. Here, we report the biochemical characterization of the direct interaction between FGF2 and Tec kinase as well as the identification of small molecules that inhibit (i) the interaction of FGF2 with Tec, (ii) tyrosine phosphorylation of FGF2 mediated by Tec in vitro and in a cellular context, and (iii) unconventional secretion of FGF2 from cells. We further demonstrate the specificity of these inhibitors for FGF2 because tyrosine phosphorylation of a different substrate of Tec is unaffected in their presence. Building on previous evidence using RNA interference, the identified compounds corroborate the role of Tec kinase in unconventional secretion of FGF2. In addition, they are valuable lead compounds with great potential for drug development aiming at the inhibition of FGF2-dependent tumor growth and metastasis.

Evidence for C-Cl/C-Br...pi interactions as an important contribution to protein-ligand binding affinity.

Attractive chlorine: Noncovalent interactions between chlorine or bromine atoms and aromatic rings in proteins open up a new method for the manipulation of molecular recognition. Substitution at distinct positions of two factor Xa inhibitors improves the free energy of binding by interaction with a tyrosine unit. The generality of this motif was underscored by multiple crystal structures as well as high-level quantum chemical calculations (see picture).

Probing the subpockets of factor Xa reveals two binding modes for inhibitors based on a 2-carboxyindole scaffold: a study combining structure-activity relationship and X-ray crystallography.

Structure-activity relationships within a series of highly potent 2-carboxyindole-based factor Xa inhibitors incorporating a neutral P1 ligand are described with particular emphasis on the structural requirements for addressing subpockets of the factor Xa enzyme. Interactions with the subpockets were probed by systematic substitution of the 2-carboxyindole scaffold, in combination with privileged P1 and P4 substituents. Combining the most favorable substituents at the indole nucleus led to the discovery of a remarkably potent factor Xa inhibitor displaying a K(i) value of 0.07 nM. X-ray crystallography of inhibitors bound to factor Xa revealed substituent-dependent switching of the inhibitor binding mode and provided a rationale for the SAR obtained. These results underscore the key role played by the P1 ligand not only in determining the binding affinity of the inhibitor by direct interaction but also in modifying the binding mode of the whole scaffold, resulting in a nonlinear SAR.

Structural requirements for factor Xa inhibition by 3-oxybenzamides with neutral P1 substituents: combining X-ray crystallography, 3D-QSAR, and tailored scoring functions.

The design, synthesis, and structure-activity relationship of 3-oxybenzamides as potent inhibitors of the coagulation protease factor Xa are described on the basis of X-ray structures, privileged structure motifs, and SAR information. A total of six X-ray structures of fXa/inhibitor complexes led us to identify the major protein-ligand interactions. The binding mode is characterized by a lipophilic dichlorophenyl substituent interacting with Tyr228 in the protease S1 pocket, while polar parts are accommodated in S4. This alignment in combination with docking allowed derivation of 3D-QSAR models and tailored scoring functions to rationalize biological affinity and provide guidelines for optimization. The resulting models showed good correlation coefficients and predictions of external test sets. Furthermore, they correspond to binding site topologies in terms of steric, electrostatic, and hydrophobic complementarity. Two approaches to derive tailored scoring functions combining binding site and ligand information led to predictive models with acceptable predictions of the external set. Good correlations to experimental affinities were obtained for both AFMoC (adaptation of fields for molecular comparison) and the novel TScore function. The SAR information from 3D-QSAR and tailored scoring functions agrees with all experimental data and provides guidelines and reasonable activity estimations for novel fXa inhibitors.

Custom chemical microarray production and affinity fingerprinting for the S1 pocket of factor VIIa.

The goal of this study was to explore the applicability of surface plasmon resonance (SPR)-based fragment screening to identify compounds that bind to factor VIIa (FVIIa). Based on pharmacophore models virtual screening approaches, we selected fragments anticipated to have a reasonable chance of binding to the S1-binding pocket of FVIIa and immobilized these compounds on microarrays. In affinity fingerprinting experiments, a number of compounds were identified to be specifically interacting with FVIIa and shown to fall into four structural classes. The results demonstrate that the chemical microarray technology platform using SPR detection generates unique chemobiological information that is useful for de novo discovery and lead development and allows the detection of weak interactions with ligands of low molecular weight.

Factor Xa inhibitors based on a 2-carboxyindole scaffold: SAR of neutral P1 substituents.

A series of novel, highly potent 2-carboxyindole-based factor Xa inhibitors is described. Structural requirements for neutral ligands, which bind in the S1 pocket of factor Xa were investigated with the 2-carboxyindole scaffold. This privileged fragment assembly approach yielded a set of equipotent, selective inhibitors with structurally diverse neutral P1 substituents.

Novel factor Xa inhibitors based on a 2-carboxyindole scaffold: SAR of P4 substituents in combination with a neutral P1 ligand.

A series of novel, highly potent 2-carboxyindole-based factor Xa inhibitors is described. Structural requirements for P4 ligands in combination with a neutral biaryl P1 ligand were investigated with the 2-carboxyindole scaffold. A diverse set of P4 substituents was identified, which, in conjunction with a biaryl P1 ligand, gave highly potent factor Xa inhibitors, which were also selective versus other proteases and efficacious in various antithrombotic secondary assays.

Novel factor Xa inhibitors based on a benzoic acid scaffold and incorporating a neutral P1 ligand.

A series of novel, highly potent, achiral factor Xa inhibitors based on a benzoic acid scaffold and containing a chlorophenethyl moiety directed towards the protease S1 pocket is described. A number of structural features, such as the requirements of the P1, P4 and ester-binding pocket ligands were explored with respect to inhibition of factor Xa. Compound 46 was found to be the most potent compound in a series of antithrombotic secondary assays.

PNA: Synthetic Polyamide Nucleic Acids with Unusual Binding Properties.

The astonishing discovery that peptide nucleic acids (PNAs, B=nucleobase), in spite of their drastic structural difference to natural DNA, are better nucleic acid mimetics than many other oligonucleotides has resulted in an explosion of research into this class of compounds. The synthesis, physical properties, and biological interactions of PNAs as well as their chimeras with DNA and RNA are summarized here.