Arylsulfanyl pyrazolones block mutant SOD1-G93A aggregation. Potential application for the treatment of amyotrophic lateral sclerosis
Graphical abstract
Introduction
Amyotrophic lateral sclerosis (ALS), also referred to as Lou Gehrig’s disease, is a neurodegenerative disease defined by both upper and lower motor neuron death, with an inexorable progression towards death within 2–5 years from diagnosis.1, 2, 3, 4 The incidence of ALS is ∼1–2/100,000/year and may be rising. Currently, there is no effective treatment for this progressive fatal disorder. Riluzole, the only FDA approved drug for the treatment of ALS, prolongs the patient life by only two to three months.5 Therefore, there is an urgent need to develop new therapies for this lethal neurodegenerative disease.
Although 10% of ALS cases are familial (FALS), the clinical and pathological features of familial and sporadic ALS are similar. This has led to a strategy using FALS mutations for elucidating disease pathogenesis and identifying potential therapeutic targets for both forms of the disease.6 Modern genetics has identified mutations in Cu/Zn superoxide dismutase 1 (SOD1) that are responsible for about 20% of the FALS patients.7, 8 Recent studies show that mutations of the TAR DNA binding protein (TDP-43)9 and the fused in sarcoma/translated in liposarcoma (FUS/TLS) gene10 also cause FALS, but at much lower percentiles than SOD1 mutations. Interestingly, abnormal TDP-43 is not present in patients with mutant SOD1 FALS, yet it is present in sporadic ALS. While the exact pathophysiological mechanisms remain unclear in sporadic ALS, it has been postulated that oxidative stress, mitochondrial impairment, glutamate excitotoxicity, defective RNA processing and transport, and aberrant protein misfolding/aggregation may lead to increasingly fragile spinal cord and cell death.1 Among these hypotheses, there is strong evidence that toxic aberrant protein misfolding/aggregation may trigger motor neuron dysfunction and cell death. Furthermore, mutant G93A SOD1 has been shown to form a distinct class of aggregated structures in PC12 cells destined for neuronal cell death.11
This PC12 cell line was previously utilized in a high-throughput screen (HTS) as a cellular model of ALS,12 which identified active compounds in the cytotoxicity assay. G93A SOD1 aggregation was cytotoxic, and inhibition of aggregation was cytoprotective. The percentage of cell survival was determined from the maximum cell viability after exposure of test compounds to G93A SOD1 aberrant protein misfolding and aggregation. The EC50 values were also determined in the protection assay from the decrease in the toxic effects of mSOD1-G93A-YFP aggregation by the treatment of test compounds. From the HTS of a 50,000-member small molecule library, arylsulfanyl pyrazolone (ASP) compounds were identified as active (Fig. 1). The hit compounds showed 100% efficacy (cell viability) compared with the positive control (radicicol, 85% efficacy) in the cytotoxicity protection assay. The most potent hit compounds produced EC50 values between 400 nM and 15 μM in the cytotoxicity protection assay (see Supplementary data). There were two distinguishing types of ASP compounds recognized from the HTS assay. Type I ASPs (Fig. 1) are 4-ethylidene-1H-pyrazol-5(4H)-ones with an alkene substituent on the pyrazolone ring; type II ASPs are 1H-pyrazol-3(2H)-ones. To initiate our optimization for this ASP scaffold, we designed and synthesized 17 ASP analogues (compound 3–19) to obtain preliminary structure–activity relationships (SAR) based on the ASP hits from the original HTS cellular model. Late drug attrition resulting from unsatisfactory pharmacokinetic (PK) profiles in clinical development has been recognized as a serious problem in the pharmaceutical industry.13, 14 Therefore, early pharmacological property assessment is crucial to successful drug development and to direct the medicinal chemistry optimization.15
While we were initiating our SAR for the potency of the ASP scaffold, we also evaluated the PK potential of this scaffold as a central nervous system (CNS) drug. Drugs targeting CNS disorders must penetrate the blood–brain barrier (BBB) to be effective; statistics show that only about 2% of potential CNS compounds can penetrate the BBB, which contributes largely to the high failure rate of CNS drug candidates.16, 17 To be a good drug candidate for the treatment of ALS, ASP derivatives must be able to demonstrate BBB permeability besides a good PK profile.18 Thus, a brain uptake study was performed as part of our early PK profile assessment of the ASP scaffold. Here we describe our efforts to improve the potency and to determine in vitro pharmacokinetic properties and ability to cross the blood–brain barrier for this ASP scaffold.
Section snippets
Chemistry
The synthesis of type I ASP compounds is outlined in Scheme 1. Furandione 20 was prepared by an acid-catalyzed aldol condensation from β-tetronic acid.19 Pyrazolone 21 was synthesized by refluxing dione 20 with hydrazine.20 The resulting alcohol (21) was treated with PBr3 to give 22; substitution of the bromine atom with thiophenol afforded 3. The synthesis of type II ASP compounds is outlined in Scheme 2. β-Ketoester 23 was prepared from ethyl 4-chloroacetoacetate and thiophenol,21 which was
Conclusion
In summary, a new chemical scaffold has been identified from a previously reported HTS cellular model targeting familial ALS. Inspired from the original ASP hits, a SAR study led to the generation of a new ASP compound (19) with improved in vitro potency of 170 nM. In vitro pharmacokinetics were carried out, and the in vivo brain/plasma ratio was determined for two ASP hit compounds (1 and 2, respectively). It appears that this scaffold needs to be modified to protect it from microsomal and
General methods
All reagents were purchased from Aldrich, Alfa Aesar, Oakwood Inc., and Maybridge Ltd. They were used without further purification unless stated otherwise. Tetrahydrofuran was distilled under nitrogen from sodium/benzophenone. Thin-layer chromatography was carried out on E. Merck precoated Silica Gel 60 F254 plates. Compounds were visualized with ferric chloride reagent or a UV lamp. Column chromatography was performed with E. Merck Silica Gel 60 (230–400 mesh). Proton nuclear magnetic
Acknowledgments
We thank the National Institutes of Health [1R43NS057849], the ALS Association (TREAT program), the Department of Defense [AL093052] and the Veterans Administration at the Edith Nourse Rogers Memorial Veterans Hospital, Bedford, MA for their generous support of the research project. The authors are grateful to Dr. Michael Avram and Lynn Luong of the Northwestern University Clinical Pharmacology Core Facility and the Pharmaceutical Chemistry Translational Resource of the Center for Molecular
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