α-Synuclein (αSN) aggregation is central to the etiology of Parkinson's disease (PD). Large-scale screening of compounds to identify aggregation inhibitors is challenged by stochastic αSN aggregation and difficulties in detecting early-stage oligomers (αSOs). We developed a high-throughput screening assay combining SDS-stimulated αSN aggregation with FRET to reproducibly detect initial stages in αSN aggregation. We screened 746,000 compounds, leading to 58 hits that markedly inhibit αSN aggregation and reduce αSOs' membrane permeabilization activity. The most effective aggregation inhibitors were derivatives of (4-hydroxynaphthalen-1-yl)sulfonamide. They interacted strongly with the N-terminal part of monomeric αSN and reduced αSO-membrane interactions, possibly by affecting electrostatic interactions. Several compounds reduced αSO toxicity toward neuronal cell lines. The inhibitors introduced chemical modifications of αSN that were, however, not a prerequisite for inhibitory activity. We also identified several phenyl-benzoxazol compounds that promoted αSN aggregation (proaggregators). These compounds may be useful tools to modulate αSN aggregation in cellula.

Piperidinyl diphenylsulfonyl sulfonamides are a novel class of molecules that have inhibitory binding affinity for sFRP-1. As a secreted protein sFRP-1 inhibits the function of the secreted Wnt glycoprotein. Therefore, as inhibitors of sFRP-1 these small molecules facilitate the Wnt/beta-catenin canonical signaling pathway. Details of the structure-activity relationships and biological activity of this structural class of compounds will be discussed.

α-synuclein is a 140-residue natively unfolded protein, whose aggregation is implicated in the development of Parkinson's Disease. It is thought that the cytotoxic species is not the mature fibril, but rather a prefibrillar aggregate which has membrane-permeabilizing properties. We have used Small Angle X-ray Scattering (SAXS) to determine the low-resolution structures of the different species formed during α-synuclein fibrillation in a non-invasive fashion. In addition to the starting monomer-dimer equilibrium and two bona fide fibril types accumulating towards the end of the aggregation process, we have identified a wreath-shaped oligomeric state which has a very distinct central hole. Both its structure and the kinetics of its formation are consistent with an on-pathway role, while its membrane-permeabilizing properties identify it as a putative cytotoxic species. We have also used SAXS to monitor the fibrillation of α-synuclein in the presence of the surfactant SDS and find that the fibrillar aggregates grow in a continuous fashion, forming beads on a string where the individual beads are stabilized by intermolecular α-synuclein contacts. The high reproducibility of this aggregative behaviour has formed the basis for a high-throughput screening assay involving 746.000 compounds that has allowed us to identify a significant number of compounds with the ability to inhibit early-stage aggregation of α-synuclein. This distinguishes the assay from previous assays that have focused mainly on the ability to prevent formation of α-synuclein fibrils. The hits from our assay may form the basis for a therapeutic intervention against Parkinson's Disease.

The naturally occurring pyranonaphthoquinone (PNQ) antibiotic lactoquinomycin and related aglycones were found to be selective inhibitors of the serine-threonine kinase AKT. A set of synthetic PNQs were prepared and a minimum active feature set and preliminary SAR were determined. PNQ lactones inhibit the proliferation of human tumor cell lines containing constitutively activated AKT and show expected effects on cellular biomarkers. Biochemical data are presented supporting a proposed bioreductive alkylation mechanism of action.

Secreted frizzled related protein-1 (sFRP-1) inhibitors have the potential to be used for the treatment of osteoporosis or other bone related disorders, since the level of sFRP-1 affects osteoblast apoptosis and proliferation. From high throughput screening, we have identified a class of iminooxothiazolidines as sFRP-1 inhibitors. Structure-activity relationships were established for various regions of the scaffold along with the biochemical characterization of this class to probe selectivity, binding and ex vivo activity. (C) 2009 Elsevier Ltd. All rights reserved.

Two classes of compounds, thiocarbamates 1 and triazoles 2, have been identified as HIV RT RNase H inhibitors using a novel FRET-based HTS assay. The potent analogs in each series exhibited selectivity and were active in cell-based assays. In addition, saturable, 1:1 stoichiometric binding to target was established and time of addition studies were consistent with inhibition of RT-mediated HIV replication.

Checkpoint deficiency of malignant cells can be exploited in cancer drug discovery. Compounds that selectively kill checkpoint-deficient cells versus checkpoint-proficient cells can be utilized to preferentially target tumor cells, while sparing normal cells. The protein p21Wafl/Cipl/Sdi1 (hereafter referred to as p21) inhibits progression of the cell cycle by inhibiting the activity of G1 kinases (cyclin D/cdk4 and cyclin E-cdk2) and the G2 kinase (cyclin B/cdkl) in response to DNA damage or abnormal DNA content. The expression of p21 is often low in human cancer cells due to frequent loss of the upstream activator, p53, and is associated with poor prognosis in some cancer patients. Using an isogenic pair of cell lines, HCT116 (p21+/+) and 80S14 (p21−/−), we have disclosed previously a novel series of pyrazolo[1,5-a]pyrimidines that preferentially kill the p21-deficient cells. We will present the synthesis, biological activities and SAR study of a series of pyrazolo[1,5-a]pyrimidines with an optimized phenyl amide moiety at the C-7 position. The mechanism of action of these compounds will also be discussed.

Canonical Wnt signaling has been demonstrated to increase bone formation, and Wnt pathway components are being pursued as potential drug targets for osteoporosis and other metabolic bone diseases, Deletion of the Writ antagonist secreted frizzled-related protein (sFRP)-1 in mice activates canonical signaling in bone and increases trabecular bone formation in aged animals. We have developed small molecules that bind to and inhibit sFRP-1 in vitro and demonstrate robust anabolic activity in an ex vivo organ culture assay. A library of over 440,000 drug-like compounds was screened for inhibitors of human sFRP-1 using a cell-based functional assay that measured activation of canonical Wnt signaling with an optimized T-cell factor (TCF)luciferase reporter gene assay. One of the hits in this screen, a diarylsulfone suifonamide, bound to sFRP-1 with a K-D of 0.35 mu M in a tryptophan fluorescence quenching assay. This Compound also selectively inhibited sFRP-1 with an EC50 of 3.9 mu M in the cell-based functional assay. Optimization of this high throughput screening hit for binding and functional potency as well as metabolic stability and other pharmaceutical properties led to improved lead compounds. One of these leads (WAY-316606) bound to sFRP-1 with a KD of 0.08 mu M and inhibited it with an EC50 of 0.65 mu M. Moreover, this compound increased total bone area in a murine calvarial organ culture assay at concentrations as low as 0.0001 mu M. This work demonstrates the feasibility of developing small molecules that inhibit sFRP-1 and stimulate canonical Wnt signaling to increase bone formation. (C) 2009 Elsevier Inc. All rights reserved.

Fluorescence polarization is a valuable technique for characterizing small molecule inhibitors. Competition fluorescence polarization assays involve the displacement of a fluorescent ligand from the enzyme with increasing amounts of a competing compound. The technique allows the determination of inhibitor dissociation constants (Ki values) and evaluation of whether the data fit is consistent with competitive and stoichiometric binding, which enables the potency of small molecule inhibitors to be ranked. Fluorescence polarization assays have the advantage of being robust, non-radioactive, and formatted for 384-well plates. Inhibitor dissociation constants can be obtained without the necessity of separating bound and unbound species. Here we describe the development and characterization of a fluorescent ligand for the Aggrecanase-1 and Aggrecanase-2 enzymes, and present measured competition IC50 values and Ki values for hit compounds in the Aggrecanase program that aided the team in lead optimization.

A series of (hetero)arylpyrimidines agonists of the Wnt-β-catenin cellular messaging system have been prepared. These compounds show activity in U2OS cells transfected with Wnt-3a, TCF-luciferase, Dkk-1 and tk-Renilla. Selected compounds show minimal GSK-3β inhibition indicating that the Wnt-β-catenin agonism activity most likely comes from interaction at Wnt-3a/Dkk-1. Two examples 1 and 25 show in vivo osteogenic activity in a mouse calvaria model. One example 1 is shown to activate non-phosphorylated β-catenin formation in bone.

The diphenylsulfonyl sulfonamide scaffold represented by 1 (WAY-316606) are small molecule inhibitors of the secreted protein sFRP-1, an endogenous antagonist of the secreted glycoprotein Wnt. Modulators of the Wnt pathway have been proposed as anabolic agents for the treatment of osteoporosis or other bone-related disorders. Details of the structure-activity relationships and biological activity from the first structural class of this scaffold will be discussed.

Inhibitor of secreted frizzled related protein-1 (sFRP-1) would be a novel potential osteogenic agent, since loss of sFRP-1 affects osteoblast proliferation, differentiation, and activity, resulting in improved bone mineral density, quality, and strength. We have identified small molecule diarylsulfone sulfonamide derivatives as sFRP-1 inhibitors. Structure-activity relationship generated for various regions of the scaffold was utilized to improve the biochemical profile, resulting in the identification of potent selective analogues, such as 16 with desirable pharmaceutical profile.

Insulin-like growth factor receptor (IGF-1R) is a growth factor receptor tyrosine kinase that acts as a critical mediator of cell proliferation and survival. Inhibitors of this receptor are believed to provide a new target in cancer therapy. We previously reported an isoquinolinedione series of IGF-1R inhibitors. Now we have identified a series of 3-cyanoquinoline compounds that are low nanomolar inhibitors of IGF-1R. The strategies, synthesis, and SAR behind the cyanoquinoline scaffold will be discussed.

Insulin-like growth factor receptor (IGF-1R) is a growth factor receptor tyrosine kinase that acts as a critical mediator of cell proliferation and survival. This receptor is over-expressed or activated in tumor cells and is emerging as a novel target in cancer therapy. Efforts in our "Hit to Lead" group have generated a novel series of submicromolar IGF-1R inhibitors based on a isoquinolinedione template originating from a Lance enzyme HTS screen. Chemical triage and parallel synthesis incorporating focused library arrays were instrumental in moving these investigations through the Wyeth exploratory medicinal chemistry process. The strategies, synthesis, and SAR behind this interesting kinase scaffold will be described.