Nuclear factor (erythroid-derived 2)-like 2 (NRF2) drug discovery: Biochemical toolbox to develop NRF2 activators by reversible binding of Kelch-like ECH-associated protein 1 (KEAP1).

Mechanisms that activate innate antioxidant responses, as a way to mitigate oxidative stress at the site of action, hold much therapeutic potential in diseases, such as Parkinson's disease, Alzheimer's disease and Huntington's disease, where the use of antioxidants as monotherapy has not yielded positive results. The nuclear factor NRF2 is a transcription factor whose activity upregulates the expression of cell detoxifying enzymes in response to oxidative stress. NRF2 levels are modulated by KEAP1, a sensor of oxidative stress. KEAP1 binds NRF2 and facilitates its ubiquitination and subsequent degradation. Recently, compounds that reversibly disrupt the NRF2-KEAP1 interaction have been described, opening the field to a new era of safer NRF2 activators. This paper describes a set of new, robust and informative biochemical assays that enable the selection and optimization of non-covalent KEAP1 binders. These include a time-resolved fluorescence resonance energy transfer (TR-FRET) primary assay with high modularity and robustness, a surface plasmon resonance (SPR) based KEAP1 direct binding assay that enables the quantification and analysis of full kinetic binding parameters and finally a 1H-15N heteronuclear single quantum coherence (HSQC) NMR assay suited to study the interaction surface of KEAP1 with residue-specific information to validate the interaction of ligands in the KEAP1 binding site.

NRF2 activation by reversible KEAP1 binding induces the antioxidant response in primary neurons and astrocytes of a Huntington's disease mouse model.

Oxidative stress has been associated with pathogenesis in several diseases including Huntington's disease (HD), a neurodegenerative disorder caused by a mutation in the huntingtin gene. Oxidative stress induced reactive oxygen species (ROS) are normally controlled at the cellular level by the nuclear factor (erythroid-derived 2)-like 2 (NRF2) a transcription factor that regulates the expression of various antioxidants and detoxifying proteins. Normally NRF2 is largely inactivated in the cytoplasm by the Kelch-like ECH-associated protein 1 (KEAP1)/Cullin-3 (CUL3) mediated ubiquitination and subsequent proteosomal degradation. In the presence of ROS, KEAP1 sensor cysteines are directly or indirectly engaged resulting in NRF2 release, nuclear translocation, and activation of its target genes. Consequently the activation of NRF2 by a small-molecule drug may have the therapeutic potential to control oxidative stress by upregulation of the endogenous antioxidant responses. Here we attempted to validate the use of a reversible non-acidic KEAP1 binder (Compound 2) to activate NRF2 with better cellular activity than similar acidic compounds. When tested head to head with sulforaphane, a covalent KEAP1 binder, Compound 2 had a similar ability to induce the expression of genes known to be modulated by NRF2 in neurons and astrocytes isolated from wild-type rat, wild type mouse and zQ175 (an HD mouse model) embryos. However, while sulforaphane also negatively affected genes involved in neurotoxicity in these cells, Compound 2 showed a clean profile suggesting its mode of action has lower off-target activity. We show that Compound 2 was able to protect cells from an oxidative insult by preserving the ATP content and the mitochondrial potential of primary astrocytes, consistent with the hypothesis that neurotoxicity induced by oxidative stress can be limited by upregulation of innate antioxidant response.

Combined Peptide and Small-Molecule Approach toward Nonacidic THIQ Inhibitors of the KEAP1/NRF2 Interaction.

The NRF2-ARE pathway is an intrinsic mechanism of defense against oxidative stress. Inhibition of the interaction between NRF2 and its main negative regulator KEAP1 is an attractive strategy toward neuroprotective agents. We report here the identification of nonacidic tetrahydroisoquinolines (THIQs) that inhibit the KEAP1/NRF2 protein-protein interaction. Peptide SAR at one residue is utilized as a tool to probe structural changes within a specific pocket of the KEAP1 binding site. We used structural information from peptide screening at the P2 pocket, noncovalent small-molecules inhibitors, and the outcome from an explorative SAR at position 5 of THIQs to identify a series of neutral THIQ analogs that bind to KEAP1 in the low micromolar range. These analogs establish new H-bond interactions at the P3 and P2 pockets allowing the replacement of the carboxylic acid functionality by a neutral primary carboxamide. X-ray crystallographic studies reveal the novel binding mode of these molecules to KEAP1.

Assay Guidance Manual: Quantitative Biology and Pharmacology in Preclinical Drug Discovery.

The Assay Guidance Manual (AGM) is an eBook of best practices for the design, development, and implementation of robust assays for early drug discovery. Initiated by pharmaceutical company scientists, the manual provides guidance for designing a "testing funnel" of assays to identify genuine hits using high-throughput screening (HTS) and advancing them through preclinical development. Combined with a workshop/tutorial component, the overall goal of the AGM is to provide a valuable resource for training translational scientists.

Polyglutamine- and temperature-dependent conformational rigidity in mutant huntingtin revealed by immunoassays and circular dichroism spectroscopy.

BACKGROUND: In Huntington's disease, expansion of a CAG triplet repeat occurs in exon 1 of the huntingtin gene (HTT), resulting in a protein bearing>35 polyglutamine residues whose N-terminal fragments display a high propensity to misfold and aggregate. Recent data demonstrate that polyglutamine expansion results in conformational changes in the huntingtin protein (HTT), which likely influence its biological and biophysical properties. Developing assays to characterize and measure these conformational changes in isolated proteins and biological samples would advance the testing of novel therapeutic approaches aimed at correcting mutant HTT misfolding. Time-resolved Förster energy transfer (TR-FRET)-based assays represent high-throughput, homogeneous, sensitive immunoassays widely employed for the quantification of proteins of interest. TR-FRET is extremely sensitive to small distances and can therefore provide conformational information based on detection of exposure and relative position of epitopes present on the target protein as recognized by selective antibodies. We have previously reported TR-FRET assays to quantify HTT proteins based on the use of antibodies specific for different amino-terminal HTT epitopes. Here, we investigate the possibility of interrogating HTT protein conformation using these assays. METHODOLOGY/PRINCIPAL FINDINGS: By performing TR-FRET measurements on the same samples (purified recombinant proteins or lysates from cells expressing HTT fragments or full length protein) at different temperatures, we have discovered a temperature-dependent, reversible, polyglutamine-dependent conformational change of wild type and expanded mutant HTT proteins. Circular dichroism spectroscopy confirms the temperature and polyglutamine-dependent change in HTT structure, revealing an effect of polyglutamine length and of temperature on the alpha-helical content of the protein. CONCLUSIONS/SIGNIFICANCE: The temperature- and polyglutamine-dependent effects observed with TR-FRET on HTT proteins represent a simple, scalable, quantitative and sensitive assay to identify genetic and pharmacological modulators of mutant HTT conformation, and potentially to assess the relevance of conformational changes during onset and progression of Huntington's disease.

Pharmacokinetics of memantine in rats and mice.

To evaluate the potential of memantine as a therapeutic agent for Huntington's disease (HD) we have undertaken a series of in vitro, ex vivo and whole animal studies to characterize its pharmacokinetics (PK) and pharmacodynamics (PD) in rats and mice. Results from these studies will enable determination of memantine exposures needed to engage the related functional PD marker and help predict the dose regimen for clinical trials to test its proposed mechanism of action; the selective blockade of extrasynaptic, but not synaptic, NMDA receptors. The studies reported here describe the PK of memantine in rats and mice at low (1 mg/kg) and high (10 mg/kg) doses. Our studies indicate that the clearance mechanisms of memantine in rats and mice are different from those in human, and that clearance needs to be taken into account when extrapolating to the human. In rats only, there is a significant metabolic contribution to memantine clearance at lower dose levels. While memantine is primarily cleared renally in all three species, the proportion of total systemic clearance above the glomerular filtration rate (GFR) is much higher in rats and mice (~13, 4.5, and 1.4 times higher than GFR in rats, mice, and humans, respectively), suggesting that the contribution of active transport to memantine elimination in rats and mice is more significant than in the human. In rats and mice, memantine had a short half-life (<4 h) and steep Cmax/Cmin ratios (>100). In the human, the half-life of memantine was reported to be very long (60-80 h) with a Cmax/Cmin ratio at steady state concentrations of ~1.5. A small change in the clearance of memantine - for example due to renal impairment or competition for the elimination pathway with a co-administered drug - will likely affect exposure and, therefore, the selectivity of memantine on NMDA receptors . The PK differences observed between these species demonstrate that the PK in mice and rats cannot be directly extrapolated to the human. Further, the relationship between the plasma concentration (and therefore dose) needed to elicit a mechanism-related in vivo functional effect (PD readout) while maintaining the selectivity of the extrasynaptic blockade of the NMDA receptors needs to be established before clinical trials can be appropriately planned.

A profiling platform for the characterization of transglutaminase 2 (TG2) inhibitors.

Huntington's disease (HD) is associated with increased expression levels and activity of tissue transglutaminase (TG2), an enzyme primarily known for its cross-linking of proteins. To validate TG2 as a therapeutic target for HD in transgenic models and for eventual clinical development, a selective and brain-permeable inhibitor is required. Here, a comprehensive profiling platform of biochemical and cellular assays is presented which has been established to evaluate the potency, cellular efficacy, subtype selectivity and the mechanism-of-action of known and novel TG2 inhibitors. Several classes of inhibitors have been characterized including: the commonly used pseudo-substrate inhibitors, cystamine and putrescine (which are generally nonspecific for TG2 and therefore not practical for drug development), the various peptidic inhibitors that target the active site cysteine residue (which display excellent selectivity but in general have poor cellular activity), and the allosteric reversible small-molecule hydrazides (which show poor selectivity and a lack of cellular activity and could not be improved despite considerable medicinal chemistry efforts). In addition, a set of inhibitors identified from a collection of pharmacologically active compounds was found to be unselective for TG2. Moreover, inhibition at the guanosine triphosphate binding site has been examined, but apart from guanine nucleotides, no such inhibitors have been identified. In addition, the promising pharmacological profile of a TG2 inhibitor is presented which is currently in lead optimization to be developed as a tool compound.