Robert Pacifici

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.

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.

Polyglutamine- and Temperature-Dependent Conformational Rigidity in Mutant Huntingtin Revealed by Immunoassays and Circular Dichroism Spectroscopy

Background In Huntingtons 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 Huntingtons disease.

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 Parkinsons disease, Alzheimers disease and Huntingtons 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.

A4 An overview of energy metabolism in huntington’s disease as a therapeutic target

Despite its well-defined causality, we still don’t know precisely how the polymorphic expansion of the CAG repeat in the first exon of the huntingtin gene results in the pleiotropic, devastating, and ultimately fatal pathophysiology of Huntington’s disease. One long-standing theory posits that the mutated protein adversely affects the cellular machinery that maintains energetic homeostasis and in so doing results in a variety of untoward downstream consequences. A large number of studies have yielded mixed results, with some supporting the ‘energetic hypothesis’ while others have not found convincing evidence for this mechanism. Two large interventional clinical trials of compounds involved in energy production (2 CARE for CoQ10 and CREST for creatine) both failed to meet their efficacy endpoints and provided no additional insights for the selection or design of subsequent energetic drug candidates. Unlike well-defined mitochondriopathies, the purported energetic changes in HD are likely to be subtle, chronic, and progressive. As such, experimental medicine studies in humans that use sensitive and specific measurements need to be conducted to build a highly detailed molecular understanding of any energetics-related deficits in HD-affected individuals. These studies will be critical to refine our mechanistic hypothesis and rationally select targets for pharmacological intervention, design shorter informative trials, and increase the confidence that future drug candidates will benefit patients. Existing data and plans for prospective studies consistent with this strategy will be presented.

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.

A14 Overview of CHDI drug development programs

CHDI is a not-for-profit drug discovery organisation that is exclusively dedicated to accelerating the development of therapeutics that slow the progression of Huntingtons disease (HD). Over the past decade we have created a large and diverse portfolio of projects to fulfil this mission. Each individual project is predicated on an evidence-based mechanistic hypothesis as to why pharmacological intervention at a particular biological target is predicted to have a beneficial effect on HD pathophysiology, and is designed to identify a proof-of-concept molecule for in vivo testing as quickly as possible. The particular therapeutic modality for each project is selected based on its relative merits, and to date have included small molecules, antibodies, antisense oligonucleotides, siRNAs, and virally-delivered genetic payloads. Our non-clinical scientists strive to optimise the drug-like properties of the candidate molecules to maximise their chances of success in the clinic. Ultimately, we believe that the target-product-profile for a drug must be informed by clinical data from the target population. Early clinical studies may be required to inform one or more of: dose (amount, frequency, and route), patient selection (disease stage), compound exposure (pharmacokinetics), target occupancy and engagement (pharmacodynamics), biological effect, safety markers, and outcomes that will be used in the larger later-stage efficacy trials. As the portfolio matures and migrates away from historical nutraceuticals and repurposed drugs to truly new-chemical-entities, we consider these issues critical to our collective success. CHDI hopes to partner with the impressive capabilities that the EHDN has built to carry out the clinical aspects of our efforts. An overview of the current programs, along with their challenges and opportunities will be presented.