Leggy A. Arnold

Enantioselective catalytic conjugate addition of dialkylzinc reagents using copper-phosphoramidite complexes; Ligand variation and non-linear effects

Abstract A variety of new chiral phosphoramidites was synthesised and tested in the copper-catalysed enantioselective conjugate addition of diethylzinc to cyclohexenone and chalcone in order to assess the structural features that are important for stereocontrol. A sterically demanding amine moiety is essential to reach high e.e.’s. Enantioselectivities for chalcones up to 89% and for cyclic enones up to 98% were found. Studies on non-linear effects with the best ligands for both cyclohexenone and chalcone showed clear non-linear effects for both cyclic and acyclic enones.

A surface on the androgen receptor that allosterically regulates coactivator binding.

Current approaches to inhibit nuclear receptor (NR) activity target the hormone binding pocket but face limitations. We have proposed that inhibitors, which bind to nuclear receptor surfaces that mediate assembly of the receptors binding partners, might overcome some of these limitations. The androgen receptor (AR) plays a central role in prostate cancer, but conventional inhibitors lose effectiveness as cancer treatments because anti-androgen resistance usually develops. We conducted functional and x-ray screens to identify compounds that bind the AR surface and block binding of coactivators for AR activation function 2 (AF-2). Four compounds that block coactivator binding in solution with IC50 ≈ 50 μM and inhibit AF-2 activity in cells were detected: three nonsteroidal antiinflammatory drugs and the thyroid hormone 3,3′,5-triiodothyroacetic acid. Although visualization of compounds at the AR surface reveals weak binding at AF-2, the most potent inhibitors bind preferentially to a previously unknown regulatory surface cleft termed binding function (BF)-3, which is a known target for mutations in prostate cancer and androgen insensitivity syndrome. X-ray structural analysis reveals that 3,3′,5-triiodothyroacetic acid binding to BF-3 remodels the adjacent interaction site AF-2 to weaken coactivator binding. Mutation of residues that form BF-3 inhibits AR function and AR AF-2 activity. We propose that BF-3 is a previously unrecognized allosteric regulatory site needed for AR activity in vivo and a possible pharmaceutical target.

Identification and characterization of the first small molecule inhibitor of MDMX.

The p53 pathway is disrupted in virtually every human tumor. In ∼50% of human cancers, the p53 gene is mutated, and in the remaining cancers, the pathway is dysregulated by genetic lesions in other genes that modulate the p53 pathway. One common mechanism for inactivation of the p53 pathway in tumors that express wild-type p53 is increased expression of MDM2 or MDMX. MDM2 and MDMX bind p53 and inhibit its function by distinct nonredundant mechanisms. Small molecule inhibitors and small peptides have been developed that bind MDM2 in the p53-binding pocket and displace the p53 protein, leading to p53-mediated cell cycle exit and apoptosis. To date, peptide inhibitors of MDMX have been developed, but no small molecule inhibitors have been reported. We have developed biochemical and cell-based assays for high throughput screening of chemical libraries to identify MDMX inhibitors and identified the first MDMX inhibitor SJ-172550. This compound binds reversibly to MDMX and effectively kills retinoblastoma cells in which the expression of MDMX is amplified. The effect of SJ-172550 is additive when combined with an MDM2 inhibitor. Results from a series of biochemical and structural modeling studies suggest that SJ-172550 binds the p53-binding pocket of MDMX, thereby displacing p53. This lead compound is a useful chemical scaffold for further optimization of MDMX inhibitors that may eventually be used to treat pediatric cancers and various adult tumors that overexpress MDMX or have similar genetic lesions. When combined with selective MDM2 inhibitors, SJ-172550 may also be useful for treating tumors that express wild-type p53.

Catalytic enantioselective annulations via 1,4-addition-aldol cyclization of functionalized organozinc reagents

Robert Naasz, Leggy A. Arnold, Mauro Pineschi,Erik Keller, and Ben L. Feringa*Department of Organic and Molecular Inorganic ChemistryUniversity of Groningen, Nijenborgh 49747 AG Groningen, The NetherlandsReceived October 23, 1998The construction of carbocyclic compounds by the Robinsonannulation is one of the essential methodologies in organic syn-thesis.

Discovery of Small Molecule Inhibitors of the Interaction of the Thyroid Hormone Receptor with Transcriptional Coregulators

Thyroid hormone (3,5,3′-triiodo-l-thyronine, T3) is an endocrine hormone that exerts homeostatic regulation of basal metabolic rate, heart rate and contractility, fat deposition, and other phenomena (1, 2). T3 binds to the thyroid hormone receptors (TRs) and controls their regulation of transcription of target genes. The binding of TRs to thyroid hormone induces a conformational change in TRs that regulates the composition of the transcriptional regulatory complex. Recruitment of the correct coregulators (CoR) is important for successful gene regulation. In principle, inhibition of the TR-CoR interaction can have a direct influence on gene transcription in the presence of thyroid hormones. Herein we report a high throughput screen for small molecules capable of inhibiting TR coactivator interactions. One class of inhibitors identified in this screen was aromatic β-aminoketones, which exhibited IC50 values of ∼2 μm. These compounds can undergo a deamination, generating unsaturated ketones capable of reacting with nucleophilic amino acids. Several experiments confirm the hypothesis that these inhibitors are covalently bound to TR. Optimization of these compounds produced leads that inhibited the TR-CoR interaction in vitro with potency of ∼0.6 μm and thyroid signaling in cellular systems. These are the first small molecules irreversibly inhibiting the coactivator binding of a nuclear receptor and suppressing its transcriptional activity.

Highly Enantioselective Copper–Phosphoramidite Catalyzed Kinetic Resolution of Chiral 2‐Cyclohexenones

A variety of substituted 2-cyclohexenones, such as (R)-1, is obtained enantiomerically pure by employing the chiral copper-phosphoramidite complex [Cu(OTf)2 L*] as a highly efficient catalyst for their kinetic resolution (>99 % ee at 52 % conversion, selectivity S>200). These important building blocks can be obtained on a synthetically interesting scale, as was demonstrated by the successful multigram resolution of 5-methyl-2-cyclohexenone. Tf=trifluoromethanesulfonyl.

Inhibition of a viral enzyme by a small-molecule dimer disruptor

Small molecule dimer disruptors that inhibit an essential dimeric protease of human Kaposi’s sarcoma-associated herpesvirus (KSHV) were identified by screening an α-helical mimetic library. Subsequently, a second generation of low micromolar inhibitors with improved potency and solubility was synthesized. Complementary methods including size exclusion chromatography and 1H-13C HSQC titration using selectively labeled 13C-Met samples revealed that monomeric protease is enriched in the presence of inhibitor. 1H-15N-HSQC titration studies mapped the inhibitor binding-site to the dimer interface, and mutagenesis studies targeting this region were consistent with a mechanism where inhibitor binding prevents dimerization through the conformational selection of a dynamic intermediate. These results validate the interface of herpesvirus proteases and other similar oligomeric interactions as suitable targets for the development of small molecule inhibitors.

Quantification of the Vitamin D Receptor - Coregulator Interaction

The vitamin D receptor (VDR) regulates a diverse set of genes that control processes including bone mineral homeostasis, immune function, and hair follicle cycling. Upon binding to its natural ligand, 1alpha,25(OH)(2)D(3), the VDR undergoes a conformational change that allows the release of corepressor proteins and the binding of coactivator proteins necessary for gene transcription. We report the first comprehensive evaluation of the interaction of the VDR with a library of coregulator binding motifs in the presence of two ligands, the natural ligand 1alpha,25(OH)(2)D(3) and a synthetic, nonsecosteroidal agonist LG190178. We show that the VDR has relatively high affinity for the second and third LxxLL motifs of SRC1, SRC2, and SRC3 and second LxxLL motif of DRIP205. This pattern is distinct in comparison to other nuclear receptors. The pattern of VDR-coregulator binding affinities was very similar for the two agonists investigated, suggesting that the biologic functions of LG190178 and 1alpha,25(OH)(2)D(3) are similar. Hairless binds the VDR in the presence of ligand through a LxxLL motif (Hr-1), repressing transcription in the presence and absence of ligand. The VDR binding patterns identified in this study may be used to predict functional differences among different tissues expressing different sets of coregulators, thus facilitating the goal of developing tissue- and gene-specific vitamin D response modulators.

Identification and analysis of hepatitis C virus NS3 helicase inhibitors using nucleic acid binding assays

Typical assays used to discover and analyze small molecules that inhibit the hepatitis C virus (HCV) NS3 helicase yield few hits and are often confounded by compound interference. Oligonucleotide binding assays are examined here as an alternative. After comparing fluorescence polarization (FP), homogeneous time-resolved fluorescence (HTRF®; Cisbio) and AlphaScreen® (Perkin Elmer) assays, an FP-based assay was chosen to screen Sigma’s Library of Pharmacologically Active Compounds (LOPAC) for compounds that inhibit NS3-DNA complex formation. Four LOPAC compounds inhibited the FP-based assay: aurintricarboxylic acid (ATA) (IC50 = 1.4 μM), suramin sodium salt (IC50 = 3.6 μM), NF 023 hydrate (IC50 = 6.2 μM) and tyrphostin AG 538 (IC50 = 3.6 μM). All but AG 538 inhibited helicase-catalyzed strand separation, and all but NF 023 inhibited replication of subgenomic HCV replicons. A counterscreen using Escherichia coli single-stranded DNA binding protein (SSB) revealed that none of the new HCV helicase inhibitors were specific for NS3h. However, when the SSB-based assay was used to analyze derivatives of another non-specific helicase inhibitor, the main component of the dye primuline, it revealed that some primuline derivatives (e.g. PubChem CID50930730) are up to 30-fold more specific for HCV NS3h than similarly potent HCV helicase inhibitors.

Improvement of Pharmacological Properties of Irreversible Thyroid Receptor Coactivator Binding Inhibitors

We have previously reported the discovery and preliminary structure activity relationships of a series of beta-aminoketones that disrupt the binding of coactivators to TR. However, the most active compounds had moderate inhibitory potency and relatively high cytotoxicity, resulting in narrow therapeutic index. Additionally, preliminary evaluation of in vivo toxicology revealed a significant dose related cardiotoxicity. Here we describe the improvement of pharmacological properties of thyroid hormone receptor coactivator binding inhibitors. A comprehensive survey of the effects of substitutents in key areas of the molecule was carried out based on mechanistic insight from the earlier report. This study revealed that both electron withdrawing and hydrophobic substituents on the aromatic ring led to higher potency. On the other hand, moving from an alkyl to a sulfonyl alkyl side chain led to reduced cytotoxicity. Finally, utilization of amine moieties having low pK(a)s resulted in lowered ion channel activity without any loss of pharmacological activity.