Matthew G. Stanton

Exploration of the internal cavity of histone deacetylase (HDAC) with selective HDAC1/HDAC2 inhibitors (SHI-1:2)

We report herein the initial exploration of novel selective HDAC1/HDAC2 inhibitors (SHI-1:2). Optimized SHI-1:2 structures exhibit enhanced intrinsic activity against HDAC1 and HDAC2, and are greater than 100-fold selective versus other HDACs, including HDAC3. Based on the SAR of these agents and our current understanding of the HDAC active site, we postulate that the SHI-1:2 extend the existing HDAC inhibitor pharmacophore to include an internal binding domain.

First Demonstration of Cerebrospinal Fluid and Plasma Aβ Lowering with Oral Administration of a β-Site Amyloid Precursor Protein-Cleaving Enzyme 1 Inhibitor in Nonhuman Primates

β-Site amyloid precursor protein (APP)-cleaving enzyme (BACE) 1 cleavage of amyloid precursor protein is an essential step in the generation of the potentially neurotoxic and amyloidogenic Aβ42 peptides in Alzheimers disease. Although previous mouse studies have shown brain Aβ lowering after BACE1 inhibition, extension of such studies to nonhuman primates or man was precluded by poor potency, brain penetration, and pharmacokinetics of available inhibitors. In this study, a novel tertiary carbinamine BACE1 inhibitor, tertiary carbinamine (TC)-1, was assessed in a unique cisterna magna ported rhesus monkey model, where the temporal dynamics of Aβ in cerebrospinal fluid (CSF) and plasma could be evaluated. TC-1, a potent inhibitor (IC50 ∼ 0.4 nM), has excellent passive membrane permeability, low susceptibility to P-glycoprotein transport, and lowered brain Aβ levels in a mouse model. Intravenous infusion of TC-1 led to a significant but transient lowering of CSF and plasma Aβ levels in conscious rhesus monkeys because it underwent CYP3A4-mediated metabolism. Oral codosing of TC-1 with ritonavir, a potent CYP3A4 inhibitor, twice daily over 3.5 days in rhesus monkeys led to sustained plasma TC-1 exposure and a significant and sustained reduction in CSF sAPPβ, Aβ40, Aβ42, and plasma Aβ40 levels. CSF Aβ42 lowering showed an EC50 of ∼20 nM with respect to the CSF [TC-1] levels, demonstrating excellent concordance with its potency in a cell-based assay. These results demonstrate the first in vivo proof of concept of CSF Aβ lowering after oral administration of a BACE1 inhibitor in a nonhuman primate.

Fluorinated piperidine acetic acids as γ-secretase modulators

We report herein a novel series of difluoropiperidine acetic acids as modulators of gamma-secretase. Synthesis of 2-aryl-3,3-difluoropiperidine analogs was facilitated by a unique and selective beta-difluorination with Selectfluor. Compounds 1f and 2c were selected for in vivo assessment and demonstrated selective lowering of Abeta42 in a genetically engineered mouse model of APP processing. Moreover, in a 7-day safety study, rats treated orally with compound 1f (250mg/kg per day, AUC(0-24)=2100microMh) did not exhibit Notch-related effects.

5′‐(E)‐Vinylphosphonate: A Stable Phosphate Mimic Can Improve the RNAi Activity of siRNA–GalNAc Conjugates

Small interfering RNA (siRNA)‐mediated silencing requires siRNA loading into the RNA‐induced silencing complex (RISC). Presence of 5′‐phosphate (5′‐P) is reported to be critical for efficient RISC loading of the antisense strand (AS) by anchoring it to the mid‐domain of the Argonaute2 (Ago2) protein. Phosphorylation of exogenous duplex siRNAs is thought to be accomplished by cytosolic Clp1 kinase. However, although extensive chemical modifications are essential for siRNA–GalNAc conjugate activity, they can significantly impair Clp1 kinase activity. Here, we further elucidated the effect of 5′‐P on the activity of siRNA–GalNAc conjugates. Our results demonstrate that a subset of sequences benefit from the presence of exogenous 5′‐P. For those that do, incorporation of 5′‐(E)‐vinylphosphonate (5′‐VP), a metabolically stable phosphate mimic, results in up to 20‐fold improved in vitro potency and up to a threefold benefit in in vivo activity by promoting Ago2 loading and enhancing metabolic stability.

Delayed and Prolonged Histone Hyperacetylation with a Selective HDAC1/HDAC2 Inhibitor.

The identification and in vitro and in vivo characterization of a potent SHI-1:2 are described. Kinetic analysis indicated that biaryl inhibitors exhibit slow binding kinetics in isolated HDAC1 and HDAC2 preparations. Delayed histone hyperacetylation and gene expression changes were also observed in cell culture, and histone acetylation was observed in vivo beyond disappearance of drug from plasma. In vivo studies further demonstrated that continuous target inhibition was well tolerated and efficacious in tumor-bearing mice, leading to tumor growth inhibition with either once-daily or intermittent administration.

Optimization of microtubule affinity regulating kinase (MARK) inhibitors with improved physical properties.

Inhibition of microtubule affinity regulating kinase (MARK) represents a potentially attractive means of arresting neurofibrillary tangle pathology in Alzheimers disease. This manuscript outlines efforts to optimize a pyrazolopyrimidine series of MARK inhibitors by focusing on improvements in potency, physical properties and attributes amenable to CNS penetration. A unique cylcyclohexyldiamine scaffold was identified that led to remarkable improvements in potency, opening up opportunities to reduce MW, Pgp efflux and improve pharmacokinetic properties while also conferring improved solubility.

Abstract 5433: Prolonged histone hyperacetylation with a novel class of HDAC1/2 selective inhibitors

The histone deacetylase (HDAC) metalloenzymes are intricately involved in gene expression through epigenetic regulation of histone acetylation. They also regulate the acetylation status of numerous non-histone proteins such as transcription factors p53, STAT1 and NF-κB as well as α-tubulin, Hsp90 and Ku70. Of the eleven zinc-dependent HDAC enzymes identified, HDACs 1 and 2 appear to be most critical in oncogenesis and tumor maintenance. They are overexpressed in many human cancers and RNAi knockdown leads to increased apoptosis. We recently disclosed a family of novel HDAC1/HDAC2-selective biaryl inhibitors. In this presentation, we will describe unique features of these biaryl inhibitors that contribute to improved preclinical efficacy and tolerability. Desirable HDAC inhibitor properties identified from preclinical experience with Zolinza™ include subtype selectivity toward HDAC1/HDAC2 and prolonged target inhibition. Compelling in vitro and in vivo data indicates that solid tumor cell lines are most sensitive to HDAC inhibition under continuous exposure rather than intermittent exposure. These biaryl inhibitors exhibit extended target engagement in vivo, and are well tolerated in nude mice. Unlike other known HDAC inhibitors, these compounds exhibit a delay in and prolongation of histone hyperacetylation in nude mice bearing HCT116 tumors, extending beyond plasma clearance of the drug. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5433.