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.

Oral delivery of diabetes peptides - Comparing standard formulations incorporating functional excipients and nanotechnologies in the translational context.

While some orally delivered diabetes peptides are moving to late development with standard formulations incorporating functional excipients, the demonstration of the value of nanotechnology in clinic is still at an early stage. The goal of this review is to compare these two drug delivery approaches from a physico-chemical and a biopharmaceutical standpoint in an attempt to define how nanotechnology-based products can be differentiated from standard oral dosage forms for oral bioavailability of diabetes peptides. Points to consider in a translational approach are outlined to seize the opportunities offered by a better understanding of both the intestinal barrier and of nano-carriers designed for oral delivery.

Sulfonylthiadiazoles with an unusual binding mode as partial dual peroxisome proliferator-activated receptor (PPAR) γ/δ agonists with high potency and in†vivo efficacy.

Compounds that simultaneously activate the peroxisome proliferator-activated receptor (PPAR) subtypes PPARγ and PPARδ have the potential to effectively target dyslipidemia and type II diabetes in a single pharmaceutically active molecule. The frequently observed side effects of selective PPARγ agonists, such as edema and weight gain, are expected to be overcome by using partial instead of full agonists for this nuclear receptor family. Herein we report the discovery, synthesis, and optimization of a novel series of sulfonylthiadiazoles that are active as partial agonists. The initial compound 6 was discovered by high-throughput screening as a moderate partial PPARδ agonist; its optimization was based on the X-ray crystal structure in complex with PPARδ. In contrast to other PPARδ agonists, this ligand does not interact directly with residues from the activation helix AF-2, which might be linked to its partial agonistic effect. Interestingly, the thiadiazole moiety fills a novel subpocket, which becomes accessible after moderate conformational rearrangement. The optimization was focused on introducing conformational constraints and replacing intramolecular hydrogen bonding interactions. Highly potent molecules with activity as dual partial PPARγ/δ agonists in the low nanomolar range were then identified. One of the most active members, compound 20 a, displayed EC50 values of 1.6 and 336 nM for PPARδ and γ, respectively. The X-ray crystal structure of its complex with PPARδ confirms our design hypothesis. Compound 20 a clearly displayed in vivo activity in two chronic mice studies. Lipids were modified in a beneficial way in normolipidemic mice, and the development of overt diabetes could be prevented in pre-diabetic db/db mice. However, body weight gain was similar to that observed with the PPARγ agonist rosiglitazone. Hence, active compounds from this series can be considered as valuable tools to elucidate the complex roles of dual PPARγ/δ agonists for potential treatment of metabolic syndrome.

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.

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.