Discovery of amido-benzisoxazoles as potent c-Kit inhibitors.

Deregulation of the receptor tyrosine kinase c-Kit is associated with an increasing number of human diseases, including certain cancers and mast cell diseases. Interference of c-Kit signaling with multi-kinase inhibitors has been shown clinically to successfully treat gastrointestinal stromal tumors and mastocytosis. Targeted therapy of c-Kit activity may provide therapeutic advantages against off-target effects for non-oncology applications. A new structural class of c-Kit inhibitors is described, including in vitro c-Kit potency, kinase selectivity, and the observed binding mode.

Automated clustering of probe molecules from solvent mapping of protein surfaces: new algorithms applied to hot-spot mapping and structure-based drug design.

Use of solvent mapping, based on multiple-copy minimization (MCM) techniques, is common in structure-based drug discovery. The minima of small-molecule probes define locations for complementary interactions within a binding pocket. Here, we present improved methods for MCM. In particular, a Jarvis-Patrick (JP) method is outlined for grouping the final locations of minimized probes into physical clusters. This algorithm has been tested through a study of protein-protein interfaces, showing the process to be robust, deterministic, and fast in the mapping of protein "hot spots." Improvements in the initial placement of probe molecules are also described. A final application to HIV-1 protease shows how our automated technique can be used to partition data too complicated to analyze by hand. These new automated methods may be easily and quickly extended to other protein systems, and our clustering methodology may be readily incorporated into other clustering packages.

Discovery of a potent and selective c-Kit inhibitor for the treatment of inflammatory diseases.

A potent and selective c-Kit inhibitor 20 was identified through a structure-activity relationship study. In an in vivo mouse model of mast cell activation, 20 blocked the SCF-induced histamine release with an EC(50) of 26 nM.

Studies toward the discovery of the next generation of antidepressants. Part 6: Dual 5-HT1A receptor and serotonin transporter affinity within a class of arylpiperazinyl-cyclohexyl indole derivatives.

Based on the previously reported discovery lead, 3-(cis-4-(4-(1H-indol-4-yl)piperazin-1-yl)cyclohexyl)-5-fluoro-1H-indole (2), a series of related arylpiperazin-4-yl-cyclohexyl indole analogs were synthesized then evaluated as 5-HT transporter inhibitors and 5-HT(1A) receptor antagonists. The investigation of the structure-activity relationships revealed the optimal pharmacophoric elements required for activities in this series. The best example from this study, 5-(piperazin-1-yl)quinoline analog (trans-20), exhibited equal binding affinities at 5-HT transporter (K(i)=4.9nM), 5-HT(1A) receptor (K(i)=6.2nM) and functioned as a 5-HT(1A) receptor antagonist.

Discovery of aryl aminoquinazoline pyridones as potent, selective, and orally efficacious inhibitors of receptor tyrosine kinase c-Kit.

Inhibition of c-Kit has the potential to treat mast cell associated fibrotic diseases. We report the discovery of several aminoquinazoline pyridones that are potent inhibitors of c-Kit with greater than 200-fold selectivity against KDR, p38, Lck, and Src. In vivo efficacy of pyridone 16 by dose-dependent inhibition of histamine release was demonstrated in a rodent pharmacodynamic model of mast cell activation.

Refining the multiple protein structure pharmacophore method: consistency across three independent HIV-1 protease models.

Developing methods to incorporate protein flexibility into structure-based drug design is an important challenge. Our approach uses multiple protein structures (MPS) to create a receptor-based pharmacophore model of the desired target. We have previously demonstrated the success of the method by applying it to human immunodeficiency virus-1 protease (HIV-1p). Our models, based on an apo structure, discriminated known HIV-1p inhibitors from druglike inactive compounds and also accurately identified bound conformations of known inhibitors. Here, we test the method by applying it to all three unbound crystal structures of HIV-1p. We have also improved our method with denser probe mapping of the binding site and refined our selection criteria for pharmacophore elements. Our improved protocol has led to the development of a consistent 8-site pharmacophore model for HIV-1p, which is independent of starting structure, and a robust MPS pharmacophore method that is more amenable to automation.

Solvation influences flap collapse in HIV-1 protease.

HIV-1 protease (HIVp) is an important target for the development of therapies to treat AIDS and is one of the classic examples of structure-based drug design. The flap region of HIVp is known to be highly flexible and undergoes a large conformational change upon binding a ligand. Accurately modeling the inherent flexibility of the HIVp system is critical for developing new methods for structure-based drug design. We report several 3-ns molecular dynamics simulations investigating the role of solvation in HIVp flap rearrangement. Using an unliganded crystal structure of HIVp, other groups have observed flap reorganization on the nanosecond timescale. We have also observed rapid, initial flap movement, but we propose that it may be caused by system setup. The initial solvation of the system creates vacuum regions around the protein that may encourage large conformational deformities. By reducing the vacuum space created by the solvation routine, the observed flap collapse is attenuated. Also, a more thorough equilibration procedure preserves a more stable protein conformation over the course of the simulation.

Incorporating protein flexibility in structure-based drug discovery: using HIV-1 protease as a test case.

We have developed a receptor-based pharmacophore method which utilizes a collection of protein structures to account for inherent protein flexibility in structure-based drug design. Several procedures were systematically evaluated to derive the most general protocol for using multiple protein structures. Most notably, incorporating more protein flexibility improved the performance of the method. The pharmacophore models successfully discriminate known inhibitors from drug-like non-inhibitors. Furthermore, the models correctly identify the bound conformations of some ligands. We used unliganded HIV-1 protease to develop and validate this method. Drug design is always initiated with a protein-ligand structure, and such success with unbound protein structures is remarkable - particularly in the case of HIV-1 protease, which has a large conformational change upon binding. This technique holds the promise of successful computer-based drug design before bound crystal structures are even discovered, which can mean a jump-start of 1-3 years in tackling some medically relevant systems with computational methods.

Development of polyphosphate parameters for use with the AMBER force field.

Accurate force fields are essential for reproducing the conformational and dynamic behavior of condensed-phase systems. The popular AMBER force field has parameters for monophosphates, but they do not extend well to polyphorylated molecules such as ADP and ATP. This work presents parameters for the partial charges, atom types, bond angles, and torsions in simple polyphosphorylated compounds. The parameters are based on molecular orbital calculations of methyldiphosphate and methyltriphosphate at the RHF/6-31+G* level. The new parameters were fit to the entire potential energy surface (not just minima) with an RMSD of 0.62 kcal/mol. This is exceptional agreement and a significant improvement over the current parameters that produce a potential surface with an RMSD of 7.8 kcal/mol to that of the ab initio calculations. Testing has shown that the parameters are transferable and capable of reproducing the gas-phase conformations of inorganic diphosphate and triphosphate. Also, the parameters are an improvement over existing parameters in the condensed phase as shown by minimizations of ATP bound in several proteins. These parameters are intended for use with the existing AMBER 94/99 force field, and they will permit users to apply AMBER to a wider variety of important enzymatic systems.

Studies toward the discovery of the next generation of antidepressants. Part 2: incorporating a 5-HT(1A) antagonist component into a class of serotonin reuptake inhibitors.

The design and synthesis of a novel series of indole derivatives (9) having dual 5-HT transporter reuptake and 5-HT(1A) antagonist activity are described.