Significance Chronic neuroinflammation and oxidative stress are likely complicit in driving disease progression in Huntington's disease (HD). Here, we describe the mechanism of action of a unique chemical scaffold that is highly selective for activation of NRF2, the master transcriptional regulator of cellular antiinflammatory and antioxidant defense genes. The use of this scaffold revealed that NRF2 activation responses were muted in HD patient-derived neural stem cells, suggesting increased susceptibility of this critical renewable cell population to oxidative stress in HD brain. However, pharmacological activation of NRF2 was able to repress inflammatory responses in mouse microglia and astrocytes, the principal cellular mediators of neuroinflammation, and in blood monocytes from HD patients. Our results suggest multiple protective benefits of NRF2 activation for HD patients.

Botulinum neurotoxins (BoNTs) are the most toxic substances known to mankind and are the causative agents of the neuroparalytic disease botulism. Their ease of production and extreme toxicity have caused these neurotoxins to be classified as Tier 1 bioterrorist threat agents and have led to a sustained effort to develop countermeasures to treat intoxication in case of a bioterrorist attack. While timely administration of an approved antitoxin is effective in reducing the severity of botulism, reversing intoxication requires different strategies. In the present study, we evaluated ABS 252 and other mercaptoacetamide small molecule active-site inhibitors of BoNT/A light chain using an integrated multi-assay approach. ABS 252 showed inhibitory activity in enzymatic, cell-based and muscle activity assays, and importantly, produced a marked delay in time-to-death in mice. The results suggest that a multi-assay approach is an effective strategy for discovery of potential BoNT therapeutic candidates.

Haspin is a serine/threonine kinase that phosphorylates Thr-3 of histone H3 in mitosis that has emerged as a possible cancer therapeutic target. High throughput screening of approximately 140,000 compounds identified the beta-carbolines harmine and harmol as moderately potent haspin kinase inhibitors. Based on information obtained from a structure-activity relationship study previously conducted for an acridine series of haspin inhibitors in conjunction with in silico docking using a recently disclosed crystal structure of the kinase, harmine analogs were designed that resulted in significantly increased haspin kinase inhibitory potency. The harmine derivatives also demonstrated less activity towards DYRK2 compared to the acridine series. In vitro mouse liver microsome stability and kinase profiling of a representative member of the harmine series (42, LDN-211898) are also presented.

LRRK2 is a large and complex protein that possesses kinase and GTPase activities and has emerged as the most relevant player in PD pathogenesis possibly through a toxic gain-of-function mechanism. Kinase activity is a critical component of LRRK2 function and represents a viable target for drug discovery. We now report the development of a mechanism-based TR-FRET assay for the LRRK2 kinase activity using full-length LRRK2. In this assay, PLK-peptide was chosen as the phosphoryl acceptor. Combination of steady state kinetic studies and computer simulations was used to calculate the initial concentrations of ATP and PLK-peptide to generate a steady state situation that favors the identification of ATP noncompetitive inhibitors. The assay was also run in the absence of GTP. Under these conditions, the assay was sensitive to inhibitors that directly interact with the kinase domain and those that modulate the kinase activity by directly interacting with other domains including the GTPase domain. The assay was optimized and used to robustly evaluate our compound library in 384-well format. An inhibitor identified through the screen was further characterized as a noncompetitive inhibitor with both ATP and PLK-peptide and showed similar inhibition against LRRK2 WT and the mutant G2019S.

This chapter contains sections titled: Importance in Drug Discovery Mechanisms of Inhibition of Two-Substrate Reactions Inhibition by Substrate Analogs Analysis of Sequential Reactions in which Inhibitor Binds to Enzyme : Product Complexes Driving SAR Programs for Two-Substrate Enzymatic Reactions