Selective LXR beta/PPAR alpha agonist improve behavioral deficits and synaptic plasticity in a model of Alzheimer’s Disease
Background and Technical innovation: Liver X Receptor, a member of the nuclear receptor family has been found to offer promising therapeutic effects for mitigating AD including reducing Aβ and improve synaptogenesis and spine growth (Figure 1). Our newest compound (AU403) is a dual nuclear receptor agonist LXR/PPAR agonist developed in silico, AU403, that is built on the advancement of a previous dual PPARδ/Ɣ agonist with the addition of LXR beta activity with minimal LXR alpha activity. In addition, we have added PPAR alpha and delta activity while removing PPARƔ activity. Thus, our design serves as a dual LXRβ - PPARδ/Ɣ- agonist. Our strategy is to avoid the unwanted side effects of traditional LXRα activity, including hepatic steatosis, elevated circulating cholesterol and neutropenia, while also avoiding PPARƔ agonism and its side effects, including edema and heart failure. Therefore, requiring lower concentrations of drug to promote protection against the observed impaired memory and pathology observed in AD. The rationale for our hypothesis is to develop 403, which is protected under a provisional patent by Auburn University, and based on the development of Dr. Amin’s previous PPAR agonist (AU9) that was patented (US 9,422,239 B1) and supported by an NIH R15AG048643 and an NIH SBIR 1R43AG065069-01 grant as a novel dual-PPAR δ/Ɣ agonist. Data to support the advancement of AU403 include: 1) intraperitoneal (IP) daily administration of AU403 in advanced (9 months) 3XTgAD (Alzheimer’s model) mice was effective at reducing AD pathology (Aβ) at low dose levels (5 mg/kg) after 1 month treatment, 2) AU403 improved neuronal plasticity (electrophysiology (LTP) studies) and memory in aged 3xTgAD mice, and 3) Developed a methyl ester formulation to improve cell permeability and brain bioavailability.
Figure 1 LXR agonism is predicted to have multiple protective mechanisms against progression of Alzheimer’s disease.
Figure 2 LXR induces cross talk between astrocytes, microglia and neurons for lipidating APOe/Aβ complexes, resulting in degradation and clearance of Aβ.
Significance of targeting LXR receptors for AD therapy. LXR receptors are a subfamily of the super family of nuclear receptors that regulate cholesterol in highly active cells in the body including the liver, brain, and heart. LXRs are regulated by oxysterols (oxidized cholesterol derivatives) as endogenous ligands for LXRs. Two isoforms of LXRs (alpha and beta), have been identified in mammals, where the LXR-β is 5 times higher in expression than LXR-α in the brain. LXRs upregulate the expression of cholesterol regulating genes including ApoE, Abca1 and Abcg1, as well as genes involved in reverse cholesterol transport (RCT), including ABC family transporters, apolipoproteins, lipoprotein remodeling enzymes, cholesterol hydroxylase, and lipogenic enzymes. It is well established that ApoE4 allele confers isoform dependent risk for late onset AD (LOAD). The apoE4 allele, also increases the probability of disease at an earlier age. In addition, apoE has been demonstrated with defining pathological lesions of Alzheimer's disease, including neurofibrillary tangles, neuritic plaques and all associated from improper cholesterol regulation and apoE lipidation process.
Figure 3 Molecular structure and ligand activation of nuclear receptors. (a) Nuclear receptors share a common structural organization with six domains (A through F): (b) Co-repressor activity without ligand LXR/RXR heterodimers in the absence of a ligand. Ligand binding results in conformational change of the heterodimer, dissociation of the corepressor complex, recruitment of co-activators, chromatin remodeling, and transcriptional activation.
Figure 4 LXR ligand binding domain. 403 differs in interactions than GW3965.
The lipidation process is critical towards the evidence describing the major effect of apoE4 on the risk of developing AD is via its effect on Aβ aggregation and clearance and influencing the onset (Figure 2). Previous LXR compounds although interact with the ligand binding domain (Figure 3) they have failed in clinical testing due to induction of hepatoxicity, elevated circulating levels of cholesterol due to strong LXRα agonism and poor brain penetrance. Our lab has computationally developed a library of hundreds of LXR compounds with variable LXR-β agonism and LXR-α interactions. We have carefully compared several computational poses of many LXR agonist in the LXR- AF2 ligand binding domain (Figure 3 and 4) and developed compounds that avoid Arg-305, and Leu-316, which are deep in the LXRα-AF2 ligand binding pocket. These sites confer full (strong) LXR-alpha agonism. Mechanistically, we further form interactions with Phe-315 which helps avoid forming bonding mostly with Arg-305 in the ligand binding domain of LXRα. AU403 was selected as our lead compound because it induces LXRβ activity (EC50 is 30nM) with minimal LXRα 800nM activity. In addition, we designed AU403 to display PPAR-delta (δ) and PPAR-alpha (α) agonism to improve anti-inflammatory activity and energy regulation in the brain, while also (PPARα) help reduce lipid accumulation in the liver and in the circulation. We recently have observed that clinically applied PPARα agonists (fenofibrate) can serve as inverse agonist for LXRα in the liver and thus prevent hepatic lipid accumulation. We have thus utilized this modeling and developed interactions with 3 Phenylalanine sites in the ligand binding domains of LXRα and observed this rational to offer significant impact on the receptor activity resulting in inverse agonism of LXRα, while conferring strong LXRb activity in the brain. Lastly, in the brain, PPARδ is the most abundant in expression of the PPAR family followed by PPAR gamma and alpha. PPARδ is known to improve energy regulation, reduce inflammation and increase fatty acid transport. While PPARα induces an increase in transport of HDL and reduces LDL levels. We are currently investigating the impact of our design on brain cholesterol formation and ability to induce communication between astrocytes and microglia via cholesterol mediated secretagogues, including cholesterol esters associated with ApoE. Thus, we have developed AU403 to follow Lipinski’s rule of 5 for improved solubility and membrane permeability, including robust LogQ (BB) values, CNS activity and MDCK cell permeability. The overall hypothesis will test that the design of AU403 allows for a safe and effective therapeutic agent for AD.
The rationale for our hypothesis is to further develop AU403, which is based upon further development of Dr. Amin’s previous PPAR agonist (AU9) that has been supported by a full patent and by an NIHR15AG048643 for a novel dual PPAR δ/Ɣ compound. Data to support the advancement of AU403 include: 1) intraperitoneal (IP) administration of AU403 in advanced (9 months) 3XTgAD (Alzheimer’s model) mice as effective for reducing AD pathology at low dose levels (5 mg/kg) after 1 month of daily treatment. 2) AU403 improves neuronal plasticity (electrophysiology studies) and impaired memory in aged 3xTgAD mice. We have received a promising score from our A1 application. Further, the program officer has asked for a 4-page response to reviewer comments and concerns. We believe this will be funded with a start date of Oct. 1, 2022. In addition, we are submitting an R01 to further explore the significance the significance of AU403 on changes in cholesterol patterns, pathology including Aβ, pTau and NFTs in 5xFAD and a TE4 animal developed by crossing P301S Tau animal with an ApoE4 knock in. Further to investigate the significance of the select amino acids in the LXR ligand binding sites by site directed substitution.
Overall Goal and Expected Outcome: The results provided from these studies will generate preclinical animal data to support an IND enabling research package to present to the biotech companies for the FDA prior to AU-403 clinical trials. We expect to identify safe and effective concentrations of AU403 in an AD mouse model and potential side effects that will help guide the future R&D strategy to be proposed in a Phase II SBIR.
Strategies and Impact: The clinical application for PPAR and LXR agonist have failed due to the chronic dosing levels required for improving the fight against AD with significant unwanted effects on human health. Our findings suggests that AU403 may serve as a preventive drug for AD. The results generated in this study will help guide the next commercialization steps to produce orally available of AU403.