Skylar J. Ferrara

Tetradentate diamino bis( thiolate) ligands (LN2S2(2-)) with saturated linkages between heteroatoms support fully reduced [(Cu(L-N2S2))2Cu2] complexes that bear relevance as an entry point toward molecules featuring the Cu2ICu2II(mu 4-S) core composition of nitrous oxide reductase (N2OR). Tetracopper [(Cu(L-N2(SMe2)2))2Cu2] (L-N2(SMe2H) 2 = N1,N2-bis(2-methyl-2mercaptopropane)-N1,N2-dimethylethane-1,2-diamine) does not support clean S atom oxidative addition but undergoes Cl atom transfer from PhICl2 or Ph3CCl to afford [(Cu(L-N2(SMe2)2))3(CuCl)5], 14. When introduced to Cu(I) sources, the L-N2(SArH) 2 ligand (LN2(SArH) 2 = N1,N2-bis(2-mercaptophenyl)-N1,N2-dimethylethane1,2-diamine), made by a newly devised route from N1,N2-bis(2fluorophenyl)-N1,N2-dimethylethane-1,2-diamine, ultimately yields the mixed-valent pentacopper [(Cu(L-N2SAr2))3Cu2] (19), which has 3-fold rotational symmetry (D3) around a Cu2 axis. The single CuII ion of 19 is ensconced within an equatorial L-N2(SAr)2(2-) ligand, as shown by 14N coupling in its EPR spectrum. Formation of 19 proceeds from an initial, fully reduced product, [(Cu(L-N2SAr2))3Cu2(Cu(MeCN))] (17), which is C2 symmetric and exceedingly air-sensitive. While unreactive toward chalcogen donors, 19 supports reversible reduction to the all-cuprous state; generation of [19]1- and treatment with S atom donors only return 19 because structural adjustments necessary for oxidative addition are noncompetitive with outer-sphere electron transfer. Oxidation of 19 is marked by intense darkening, consistent with greater mixed valency, and by dimerization in the crystalline state to a decacopper species ([20]2+) of S4 symmetry.

Triggering receptor expressed on myeloid cells-2 (TREM2) is a cell surface receptor on macrophages and microglia that senses and responds to disease associated signals to regulate the phenotype of these innate immune cells. The TREM2 signaling pathway has been implicated in a variety of diseases ranging from neurodegeneration in the central nervous system to metabolic disease in the periphery. We report here that TREM2 is a thyroid hormone regulated gene and its expression in macrophages and microglia is stimulated by thyroid hormone. Both endogenous thyroid hormone and sobetirome, a synthetic thyroid hormone agonist drug, suppress pro-inflammatory cytokine production from myeloid cells including macrophages that have been treated with the SARS-CoV-2 spike protein which produces a strong, pro-inflammatory phenotype. Thyroid hormone agonism was also found to induce phagocytic behavior in microglia, a phenotype consistent with activation of the TREM2 pathway. The thyroid hormone antagonist NH-3 blocks the anti-inflammatory effects of thyroid hormone agonists and suppresses microglia phagocytosis. Finally, in a murine experimental autoimmune encephalomyelitis (EAE) multiple sclerosis model, treatment with Sob-AM2, a CNS-penetrating sobetirome prodrug, results in increased Trem2 expression in disease lesion resident myeloid cells which correlates with therapeutic benefit in the EAE clinical score and reduced damage to myelin. Our findings represent the first report of endocrine regulation of TREM2 and provide a unique opportunity to drug the TREM2 signaling pathway with orally active small molecule therapeutic agents.

The blood-brain barrier is a major impediment for targeted central nervous system (CNS) therapeutics, especially with carboxylic acid-containing drugs. Nuclear receptors modulators, which often feature carboxylic acid motifs for target engagement, have emerged as a class of potentially powerful therapeutics for neurodegenerative CNS diseases. Herein is described a prodrug strategy which directs the biodistribution of parent drug nuclear receptor modulators into the CNS while masking them as functional receptor ligands in the periphery. This prodrug strategy targets a specific amidase, fatty-acid amide hydrolase (FAAH), an enzyme with enriched expression in the CNS. Our results demonstrate that this prodrug strategy can be generalized to a variety of carboxylic acid-containing drug structures that satisfy the structural requirements of blood-brain barrier diffusion and FAAH substrate recognition.

Neurodegenerative diseases lack a breadth of treatment options due, in part, to the known difficulties associated with a drug’s passage across the blood-brain barrier. In this study, we provide a blueprint for how to get drug-like structures which feature carboxylic acid motifs across the blood-brain barrier, while attenuating their peripheral exposure using a chemical prodrug strategy. This prodrug strategy utilizes endogenous fatty-acid amide hydrolase expressed in the CNS to cleave and release parent drugs for CNS drug action to commence. In particular, we demonstrate the successes and limitations of this prodrug strategy within a series of nuclear receptor modulators, which have shown promise as potential therapeutics for neurodegenerative diseases.

Oligodendrocyte processes wrap axons to form neuroprotective myelin sheaths, and damage to myelin in disorders, such as multiple sclerosis (MS), leads to neurodegeneration and disability. There are currently no approved treatments for MS that stimulate myelin repair. During development, thyroid hormone (TH) promotes myelination through enhancing oligodendrocyte differentiation; however, TH itself is unsuitable as a remyelination therapy due to adverse systemic effects. This problem is overcome with selective TH agonists, sobetirome and a CNS-selective prodrug of sobetirome called Sob-AM2. We show here that TH and sobetirome stimulated remyelination in standard gliotoxin models of demyelination. We then utilized a genetic mouse model of demyelination and remyelination, in which we employed motor function tests, histology, and MRI to demonstrate that chronic treatment with sobetirome or Sob-AM2 leads to significant improvement in both clinical signs and remyelination. In contrast, chronic treatment with TH in this model inhibited the endogenous myelin repair and exacerbated disease. These results support the clinical investigation of selective CNS-penetrating TH agonists, but not TH, for myelin repair.

Thyroid hormone (TH) action is of clinical interest in treating demyelinating diseases of the central nervous system (CNS). Two amide prodrugs of sobetirome, a potent thyroid hormone agonist, were previously shown to significantly improve CNS selectivity of the parent drug through hydrolysis by fatty-acid amide hydrolase (FAAH). This concept is elaborated upon here with a series of 29 amide prodrugs targeting FAAH. We identify that conservative aliphatic modifications such as the N-methyl (4), N-ethyl (5), N-fluoroethyl (15), and N-cyclopropyl (18) substantially favor selective CNS distribution of the parent drug in mice. Additionally, lead compounds exhibit moderate to good rates of hydrolysis at FAAH in vitro suggest-ing both enzymatic and physicochemical properties are important parameters for optimization. Both 4 and 15 were orally bioavailable while retaining appreciable CNS selectivity following an oral dose. The pharmacokinetic parameters of 4 over 24 hours post dose (i.v. and p.o.) were determined.

Thyromimetics represent a class of experimental drugs that can stimulate tissue-selective thyroid hormone action. As such, thyromimetics should have effects on the hypothalamic-pituitary-thyroid (HPT) axis, but details of this action and the subsequent effects on systemic thyroid hormone levels have not been reported to date. Here, we compare the HPT-axis effects of sobetirome, a well-studied thyromimetic, with Sob-AM2, a newly developed prodrug of sobetirome that targets sobetirome distribution to the central nervous system (CNS). Similar to endogenous thyroid hormone, administration of sobetirome and Sob-AM2 suppress HPT-axis gene transcript levels in a manner that correlates to their specific tissue distribution properties (periphery vs CNS, respectively). Dosing male C57BL/6 mice with sobetirome and Sob-AM2 at concentrations ≥10 μg/kg/d for 29 days induces a state similar to central hypothyroidism characterized by depleted circulating T4 and T3 and normal TSH levels. However, despite the systemic T4 and T3 depletion, the sobetirome- and Sob-AM2-treated mice do not show signs of hypothyroidism, which may result from the presence of the thyromimetic in the thyroid hormone–depleted background.

Thyroid hormone is a principal regulator of essential processes in vertebrate physiology and homeostasis. Synthetic derivatives of thyroid hormone, known as thyromimetics, display desirable therapeutic properties. Thoroughly understanding how thyromimetics distribute throughout the body is crucial for their development and this requires appropriate bioanalytical techniques to quantify drug levels in different tissues. Here, we describe a detailed protocol for the quantification of the thyromimetic sobetirome using liquid chromatography tandem-mass spectrometry (LC-MS/MS).

The blood–brain barrier (BBB) can be a substantial impediment to achieving therapeutic levels of drugs in the CNS. Certain chemical functionality such as the carboxylic acid is a general liability for BBB permeability preventing significant CNS distribution of a drug from a systemic dose. Here, we report a strategy for CNS-selective distribution of the carboxylic acid containing thyromimetic sobetirome using prodrugs targeted to fatty-acid amide hydrolase (FAAH), which is expressed in the brain. Two amide prodrugs of sobetirome were shown to be efficient substrates of FAAH with V_max/K_M values comparable to the natural endocannabinoid FAAH substrate anandamide. In mice, a systemic dose of sobetirome prodrug leads to a substantial ∼60-fold increase in brain distribution (K_p) of sobetirome compared to an equimolar systemic dose of the parent drug. The increased delivery of sobetirome to the brain from the prodrug was diminished by both pharmacological inhibition and genetic deletion of FAAH in vivo. The increased brain exposure of sobetirome arising from the prodrug corresponds to ∼30-fold increased potency in brain target engagement compared to the parent drug. These results suggest that FAAH-targeted prodrugs can considerably increase drug exposure to the CNS with a concomitant decrease in systemic drug levels generating a desirable distribution profile for CNS acting drugs.