SUMMARY The neurotransmitter dopamine (DA) is important for numerous biological functions, including control of movement. Oxidation of DA to highly toxic and reactive species has been hypothesized to contribute to the selective neurodegeneration observed in Parkinson's disease (PD). DA catabolism is initiated by oxidative deamination via monoamine oxidase to yield 3,4-dihydroxyphenylacetaldehyde (DOPAL). Such metabolism can be problematic as it greatly increases the toxicity of DA by production of DOPAL, known to be a toxic and reactive intermediate. DOPAL undergoes carbonyl metabolism primarily via aldehyde dehydrogenase (ALDH) enzymes to a less toxic acid product. Previous studies from our laboratory have shown that cellular ALDH enzymes are sensitive towards products of oxidative stress and lipid peroxidation, which are thought to be elevated during PD pathogenesis. Inhibition of ALDH and the resulting accumulation of DOPAL are concerning as DOPAL is toxic to dopaminergic cells, readily modifies proteins and causes protein aggregation. In addition, pesticides with association between exposure and PD incidence can interfere with DA metabolism and trafficking and/or ALDH activity, directly or indirectly, yielding elevation of DOPAL. Therefore, impairment of carbonyl metabolism is a potential mechanistic link between cellular insult and generation of a toxic and reactive intermediate endogenous to dopamine neurons.

3,4-Dihydroxyphenylacetaldehyde (DOPAL) is an endogenously produced toxic aldehyde. It is a bifunctional electrophile implicated in the loss of dopaminergic cells concomitant with Parkinson’s disease and neurodegeneration. DOPAL is known to react with proteins and amino acids such as N-acetyl Lys; oxidation of the catechol moiety to the quinone of DOPAL increases this reactivity. Here we demonstrate the ability of the antioxidants N-acetylcysteine, glutathione, and ascorbic acid to mitigate the reactivity of DOPAL with proteins and amino acids in a dose-dependent fashion. Conversely, Trolox did not lessen the observed reactivity with proteins. Interestingly, use of tricine, a buffer and reducing agent, in these systems also decreased the reactivity of DOPAL with amines, yielding tricine-derived free radical species. Modification of amines with aldehydes typically involves Schiff base chemistry; however, the observance of free radicals suggests that an oxidative step is involved in the reaction of DOPAL with lysine. Furthermore, while Schiff base formation is usually optimal at pH 5, the reaction rate of DOPAL with N-acetyl Lys is negligible at pH 5 and is enhanced under basic conditions (e.g. pH 9). Conditions of high pH are also favorable for catechol auto-oxidation, known to occur for DOPAL. The antioxidant-mediated protection demonstrated here suggests that oxidative stress may impart cellular vulnerability to protein modification by DOPAL. Therefore, depleted antioxidants and increased levels of lipid peroxidation products, known to prevent detoxifying metabolism of DOPAL, may present a survival challenge to dopaminergic cells targeted in Parkinson’s disease.

Regulator of G Protein Signaling (RGS) 17 is an overexpressed promoter of cancer survival in lung and prostate tumors, the knockdown of which results in decreased tumor cell proliferation in vitro. Identification of drug-like molecules inhibiting this protein could ameliorate the RGS17’s pro-tumorigenic effect. Using high-throughput screening, a chemical library containing natural products was interrogated for inhibition of the RGS17–Gαo interaction. Initial hits were verified in control and counter screens. Leads were characterized via biochemical, mass spectrometric, Western blot, microscopic, and cytotoxicity measures. Four known compounds (1–4) were identified with IC50 values ranging from high nanomolar to low micromolar. Three compounds were extensively characterized biologically, demonstrating cellular activity determined by confocal microscopy, and two compounds were assessed via ITC exhibiting high nanomolar to low micromolar dissociation constants. The compounds were found to have a cysteine-dependent mechanism of binding, verified through site-directed mutagenesis and cysteine reactivity assessment. Two compounds, sanguinarine (1) and celastrol (2), were found to be cytostatic against lung and prostate cancer cell lines and cytotoxic against prostate cancer cell lines in vitro, although the dependence of RGS17 on these phenomena remains elusive, a result that is perhaps not surprising given the multimodal cytostatic and cytotoxic activities of many natural products.

Regulator of G protein signaling (RGS) proteins temporally regulate heterotrimeric G protein signaling cascades elicited by G protein-coupled receptor activation and thus are essential for cell homeostasis. The dysregulation of RGS protein expression has been linked to several pathologies, spurring discovery efforts to identify small-molecule inhibitors of these proteins. Presented here are the results of a high-throughput screening (HTS) campaign targeting RGS17, an RGS protein reported to be inappropriately upregulated in several cancers. A screen of over 60,000 small molecules led to the identification of five hit compounds that inhibit the RGS17-Gα protein-protein interaction. Chemical and biochemical characterization demonstrated that three of these hits inhibited the interaction through the decomposition of parent compound into reactive products under normal chemical library storage/usage conditions. Compound substructures susceptible to decomposition are reported and the decomposition process characterized, adding to the armamentarium of tools available to the screening field, allowing for the conservation of resources in follow-up efforts and more efficient identification of potentially decomposed compounds. Finally, analogues of one hit compound were tested, and the results establish the first ever structure-activity relationship (SAR) profile for a small-molecule inhibitor of RGS17.

Dopamine (DA) is a monoamine neurotransmitter important for the control of movement. When not sequestered in vesicles, DA is metabolized by monoamine oxidase (MAO) to 3,4-dihydroxyphenylacetaldehyde (DOPAL), which can further undergo metabolism by aldehyde dehydrogenases (ALDH) and aldehyde/aldose reductases (AR) to 3,4-dihydroxyphenylacetic acid, and 3,4-dihydroxyphenylethanol, respectively. Other monoamines such as norepinephrine and serotonin also are metabolized by MAO, yielding biogenic aldehydes. DOPAL has been shown to be toxic to dopaminergic cells at levels close to those measured for physiologic conditions. Generation of the DA-derived aldehyde at aberrant levels could be due to several mechanisms, including: inhibition of ALDH and AR via oxidative stress products, impairment of DA trafficking, and direct inhibition of carbonyl metabolism. Models of altered DA metabolism or trafficking demonstrate toxic endpoints and/or progressive loss of DA neurons, indicating that proper DA metabolism and trafficking is critical for health of DA neurons. DOPAL is a highly reactive aldehyde intermediate that modifies proteins and causes protein aggregation. In addition, the DA-derived aldehyde can auto-oxidize to a quinone, producing reactive oxygen species (ROS), and generates ROS during the reaction with proteins. Given such evidence, DOPAL is hypothesized to be an endogenous neurotoxin and mechanistic link between exposure or insults and conditions relevant to neurodegenerative disease.