Methyl-substitution of an iminohydantoin spiropiperidine ß-secretase (BACE-1) inhibitor has a profound effect on its potency.

The IC50 of a beta-secretase (BACE-1) lead compound was improved ∼200-fold from 11 µM to 55 nM through the addition of a single methyl group. Computational chemistry, small molecule NMR, and protein crystallography capabilities were used to compare the solution conformation of the ligand under varying pH conditions to its conformation when bound in the active site. Chemical modification then explored available binding pockets adjacent to the ligand. A strategically placed methyl group not only maintained the required pKa of the piperidine nitrogen and filled a small hydrophobic pocket, but more importantly, stabilized the conformation best suited for optimized binding to the receptor.

Total synthesis of aeruginosin 98B.

The first total synthesis of aeruginosin 98B was accomplished. The key step is a highly diastereoselective Pd-catalyzed intramolecular asymmetric allylic alkylation reaction of a diastereomeric mixture of allylic carbonates that is enabled by the use of racemic phosphine ligand L1.

Fragment-based discovery of nonpeptidic BACE-1 inhibitors using tethering.

BACE-1 (beta-site amyloid precursor protein cleaving enzyme), a prominent target in Alzheimer's disease drug discovery efforts, was surveyed using Tethering technology to discover small molecule fragment ligands that bind to the enzyme active site. Screens of a library of >15000 thiol-containing fragments versus a panel of BACE-1 active site cysteine mutants under redox-controlled conditions revealed several novel amine-containing fragments that could be selectively captured by subsets of the tethering sites. For one such hit class, defined by a central aminobenzylpiperidine (ABP) moiety, X-ray crystal structures of BACE mutant-disulfide conjugates revealed that the fragment bound by engaging both catalytic aspartates with hydrogen bonds. The affinities of ABP fragments were improved by structure-guided chemistry, first for conjugation as thiol-containing fragments and then for stand-alone, noncovalent inhibition of wild-type (WT) BACE-1 activity. Crystallography confirmed that the inhibitors bound in exactly the same mode as the disulfide-conjugated fragments that were originally selected from the screen. The ABP ligands represent a new type of nonpeptidic BACE-1 inhibitor motif that has not been described in the aspartyl protease literature and may serve as a starting point for the development of BACE-1-directed Alzheimer's disease therapeutics.

Modifications of the isonipecotic acid fragment of SNS-032: analogs with improved permeability and lower efflux ratio.

Modifications of the isonipecotic acid fragment of SNS-032 results in analogs which are more permeable and lower effluxed than SNS-032. The enantiomerically pure synthesis and the in vivo profile of analog 20 is described.

A diaminocyclohexyl analog of SNS-032 with improved permeability and bioavailability properties.

The identification of a selective CDK2, 7, 9 inhibitor 4 with improved permeability is described. Compound 4 exhibits comparable CDK selectivity profile to SNS-032, but shows improved permeability and higher bioavailability in mice.

Design and synthesis of 2-amino-pyrazolopyridines as Polo-like kinase 1 inhibitors.

A series of 2-amino-pyrazolopyridines was designed and synthesized as Polo-like kinase (Plk) inhibitors based on a low micromolar hit. The SAR was developed to provide compounds exhibiting low nanomolar inhibitory activity of Plk1; the phenotype of treated cells is consistent with Plk1 inhibition. A co-crystal structure of one of these compounds with zPlk1 confirms an ATP-competitive binding mode.

Tethering identifies fragment that yields potent inhibitors of human caspase-1.

Disulfide Tethering was applied to the active site of human caspase-1, resulting in the discovery of a novel, tricyclic molecular fragment that selectively binds in S4. This fragment was developed into a class of potent inhibitors of human caspase-1. Several key analogues determined the optimal distance of the tricycle from the catalytic residues, the relative importance of various features of the tricycle, and the importance of the linker.

Structural analysis of caspase-1 inhibitors derived from Tethering.

Caspase-1 is a key endopeptidase responsible for the post-translational processing of the IL-1beta and IL-18 cytokines and small-molecule inhibitors that modulate the activity of this enzyme are predicted to be important therapeutic treatments for many inflammatory diseases. A fragment-assembly approach, accompanied by structural analysis, was employed to generate caspase-1 inhibitors. With the aid of Tethering with extenders (small molecules that bind to the active-site cysteine and contain a free thiol), two novel fragments that bound to the active site and made a disulfide bond with the extender were identified by mass spectrometry. Direct linking of each fragment to the extender generated submicromolar reversible inhibitors that significantly reduced secretion of IL-1beta but not IL-6 from human peripheral blood mononuclear cells. Thus, Tethering with extenders facilitated rapid identification and synthesis of caspase-1 inhibitors with cell-based activity and subsequent structural analyses provided insights into the enzyme's ability to accommodate different inhibitor-binding modes in the active site.

Allosteric inhibition of protein tyrosine phosphatase 1B.

Obesity and type II diabetes are closely linked metabolic syndromes that afflict >100 million people worldwide. Although protein tyrosine phosphatase 1B (PTP1B) has emerged as a promising target for the treatment of both syndromes, the discovery of pharmaceutically acceptable inhibitors that bind at the active site remains a substantial challenge. Here we describe the discovery of an allosteric site in PTP1B. Crystal structures of PTP1B in complex with allosteric inhibitors reveal a novel site located approximately 20 A from the catalytic site. We show that allosteric inhibitors prevent formation of the active form of the enzyme by blocking mobility of the catalytic loop, thereby exploiting a general mechanism used by tyrosine phosphatases. Notably, these inhibitors exhibit selectivity for PTP1B and enhance insulin signaling in cells. Allosteric inhibition is a promising strategy for targeting PTP1B and constitutes a mechanism that may be applicable to other tyrosine phosphatases.

Identification of potent and novel small-molecule inhibitors of caspase-3.

The design and synthesis of a series of novel, reversible, small molecule inhibitors of caspase-3 are described.

Identification of potent and selective small-molecule inhibitors of caspase-3 through the use of extended tethering and structure-based drug design.

The design, synthesis, and in vitro activities of a series of potent and selective small-molecule inhibitors of caspase-3 are described. From extended tethering, a salicylic acid fragment was identified as having binding affinity for the S(4) pocket of caspase-3. X-ray crystallography and molecular modeling of the initial tethering hit resulted in the synthesis of 4, which reversibly inhibited caspase-3 with a K(i) = 40 nM. Further optimization led to the identification of a series of potent and selective inhibitors with K(i) values in the 20-50 nM range. One of the most potent compounds in this series, 66b, inhibited caspase-3 with a K(i) = 20 nM and selectivity of 8-500-fold for caspase-3 vs a panel of seven caspases (1, 2, and 4-8). A high-resolution X-ray cocrystal structure of 4 and 66b supports the predicted binding modes of our compounds with caspase-3.