Targeting the Ets Binding Site of the HER2/neu Promoter with Pyrrole-Imidazole Polyamides

Three DNA binding polyamides (1-3) were synthesized that bind with high affinity (Ka = 8.7·10^9 M^-1 to 1.4·10^10 M^-1) to two 7-base pair sequences overlapping the Ets DNA binding site (EBS; GAGGAA) within the regulatory region of the HER2/neu proximal promoter. As measured by electrophoretic mobility shift assay, polyamides binding to flanking elements upstream (1) or downstream (2 and 3) of the EBS were one to two orders of magnitude more effective than the natural product distamycin at inhibiting formation of complexes between the purified EBS protein, epithelial restricted with serine box (ESX), and the HER2/neu promoter probe. One polyamide, 2, completely blocked Ets-DNA complex formation at 10 nM ligand concentration, whereas formation of activator protein-2-DNA complexes was unaffected at the activator protein-2 binding site immediately upstream of the HER2/neu EBS, even at 100 nM ligand concentration. At equilibrium, polyamide 1 was equally effective at inhibiting Ets/DNA binding when added before or after in vitro formation of protein-promoter complexes, demonstrating its utility to disrupt endogenous Ets-mediated HER2/neu preinitiation complexes. Polyamide 2, the most potent inhibitor of Ets-DNA complex formation by electrophoretic mobility shift assay, was also the most effective inhibitor of HER2/neu promoter-driven transcription measured in a cell-free system using nuclear extract from an ESX- and HER2/neu-overexpressing human breast cancer cell line, SKBR-3

Chorea-related mutations in PDE10A result in aberrant compartmentalization and functionality of the enzyme

A robust body of evidence supports the concept that phosphodiesterase 10A (PDE10A) activity in the basal ganglia orchestrates the control of coordinated movement in human subjects. Although human mutations in the PDE10A gene manifest in hyperkinetic movement disorders that phenocopy many features of early Huntington’s disease, characterization of the maladapted molecular mechanisms and aberrant signaling processes that underpin these conditions remains scarce. Recessive mutations in the GAF-A domain have been shown to impair PDE10A function due to the loss of striatal PDE10A protein levels, but here we show that this paucity is caused by irregular intracellular trafficking and increased PDE10A degradation in the cytosolic compartment. In contrast to GAF-A mutants, dominant mutations in the GAF-B domain of PDE10A induce PDE10A misfolding, a common pathological phenotype in many neurodegenerative diseases. These data demonstrate that the function of striatal PDE10A is compromised in disorders where disease-associated mutations trigger a reduction in the fidelity of PDE compartmentalization

A comparative study of fragment screening methods on the p38α kinase: new methods, new insights

The stress-activated kinase p38α was used to evaluate a fragment-based drug discovery approach using the BioFocus fragment library. Compounds were screened by surface plasmon resonance (SPR) on a Biacore(™) T100 against p38α and two selectivity targets. A sub-set of our library was the focus of detailed follow-up analyses that included hit confirmation, affinity determination on 24 confirmed, selective hits and competition assays of these hits with respect to a known ATP binding site inhibitor. In addition, functional activity against p38α was assessed in a biochemical assay using a mobility shift platform (LC3000, Caliper LifeSciences). A selection of fragments was also evaluated using fluorescence lifetime (FLEXYTE(™)) and microscale thermophoresis (Nanotemper) technologies. A good correlation between the data for the different assays was found. Crystal structures were solved for four of the small molecules complexed to p38α. Interestingly, as determined both by X-ray analysis and SPR competition experiments, three of the complexes involved the fragment at the ATP binding site, while the fourth compound bound in a distal site that may offer potential as a novel drug target site. A first round of optimization around the remotely bound fragment has led to the identification of a series of triazole-containing compounds. This approach could form the basis for developing novel and active p38α inhibitors. More broadly, it illustrates the power of combining a range of biophysical and biochemical techniques to the discovery of fragments that facilitate the development of novel modulators of kinase and other drug targets. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10822-011-9454-9) contains supplementary material, which is available to authorized users

Sequence-specific DNA recognition by polyamides

Sequence-specific DNA-binding small molecules that can permeate cells could potentially regulate transcription of specific genes. Simple pairing rules for the minor groove of the double helix have been developed that allow the design of ligands for predetermined DNA sequences. Some of these polyamides have been shown to inhibit specific gene expression in mammalian cell culture

Compounds activating VCP D1 ATPase enhance both autophagic and proteasomal neurotoxic protein clearance.

Enhancing the removal of aggregate-prone toxic proteins is a rational therapeutic strategy for a number of neurodegenerative diseases, especially Huntington's disease and various spinocerebellar ataxias. Ideally, such approaches should preferentially clear the mutant/misfolded species, while having minimal impact on the stability of wild-type/normally-folded proteins. Furthermore, activation of both ubiquitin-proteasome and autophagy-lysosome routes may be advantageous, as this would allow effective clearance of both monomeric and oligomeric species, the latter which are inaccessible to the proteasome. Here we find that compounds that activate the D1 ATPase activity of VCP/p97 fulfill these requirements. Such effects are seen with small molecule VCP activators like SMER28, which activate autophagosome biogenesis by enhancing interactions of PI3K complex components to increase PI(3)P production, and also accelerate VCP-dependent proteasomal clearance of such substrates. Thus, this mode of VCP activation may be a very attractive target for many neurodegenerative diseases.We are grateful for funding from the UK Dementia Research Institute (funded by the MRC, Alzheimer’s Research UK and the Alzheimer’s Society) (to DCR), The Tau Consortium, Alzheimer’s Research UK, an anonymous donation to the Cambridge Centre for Parkinson-Plus, AstraZeneca, the Swedish Natural Research Council (VR) (to S.M.H; reference 2016–06605) and from the European Molecular Biology Organisation (EMBO long-term fellowships to SMH and LW; ALTF 1024-2016 and ALTF 135-2016, respectively)

An efficient and scalable process to produce morpholine-d₈

<p>Incorporation of isotopes has long been used as a research tool to label carbons and elucidate biochemical pathways. More recently, H→D exchange has led to analogs of therapeutic agents with improved metabolic stability and properties. Such compounds also have the potential for an improved drug/drug interaction profile and may even avoid the formation of toxic metabolites. Hence, a clear need for an efficient access to deuterated intermediates on large scale has emerged. In the context of an ongoing drug discovery program, we required large quantities of morpholine-d₈. We herein report the successful optimization of a one-pot process allowing a near complete exchange of all methylene hydrogens in morpholine to deuterium atoms using D₂O as the sole source of deuterium and Raney Nickel as catalyst. This facile and safe protocol will be used to scale up the synthesis of morpholine-d₈ in due course.</p

Brain-to-plasma concentration ratio (B:P) following a single SC or PO administration to mice.

<p>NC = not calculated since concentrations were below the lower limit of assay quantitation (3 nM).</p>(a)<p>Values represent mean (standard deviation); N = 3.</p

Biochemical and Cellular HDAC inhibition by 4b.

<p>(A) % inhibition of human recombinant Class I enzymes HDAC1 (red), HDAC2 (green), HDAC3 (black) and HDAC8 (blue) by <b>4b</b>. (B) No inhibition of ClassIIa/b enzymes by <b>4b</b>; HDAC 4(green), HDAC5 (red), HDAC7 (purple), HDAC9 (orange), HDAC6 (brown). (C) Time-dependence of human recombinant HDAC3 inhibition by varying preincubation time of <b>4b</b> with enzyme (as shown). (D) Cellular inhibition of endogenous Class I HDACs/HDAC6 using Boc_Lys_Ac (black traces) or Class IIa/HDAC8 HDACs using Boc_Lys_TFA substrate (red traces) by <b>4b</b> (closed circles) or reference compounds SAHA and Compound 26. (E) Time- dependence of cellular Class I HDAC inhibition by varying preincubation of <b>4b</b> with cells (as shown). (F) Plot of IC₅₀ values versus compound-cell preincubation time for SAHA (green) and <b>4b</b> (black).</p