Thatcher, G. R. | Search bioRxiv

bioRxiv2025

In search of nonlipogenic ABCA1 inducers (NLAI): precision coregulator TR-FRET identifies diverse signatures for LXR ligands

APOE4, the major genetic risk factor for Alzheimers disease (AD), and ABCA1, required for lipidation of APOE are gene products of the liver X receptor (LXR) receptor. LXR agonists have been validated in animal models as therapeutics for AD, atherosclerosis, and many other diseases. Clinical progress has been thwarted by unwanted hepatic lipogenesis. Structurally diverse LXR ligands were profiled in coregulator TR-FRET (CRT) assays analyzing ligand-induced coactivator recruitment, coactivator selectivity, corepressor dissociation, and LXR isoform selectivity. A multiplex CRT assay was developed to measure synchronous ligand-induced displacement of corepressor by coactivator. Potency for coactivator recruitment to LXR{beta} correlated with induction of ABCA1 in human astrocytoma cells. Correlation with lipogenic activation of sterol response element (SRE) in hepatocarcinoma cells, was more complex. CRT response was diverse revealing ligands with theoretical full agonist, partial agonist, antagonist, and inverse agonist, and other signatures within the same chemical series, suggesting the scope for precision CRT to guide nonlipogenic LXR agonist design.

pharmacology and toxicology↗

bioRxiv2021

Potent, Novel SARS-CoV-2 PLpro Inhibitors Block Viral Replication in Monkey and Human Cell Cultures

Antiviral agents blocking SARS-CoV-2 viral replication are desperately needed to complement vaccination to end the COVID-19 pandemic. Viral replication and assembly are entirely dependent on two viral cysteine proteases: 3C-like protease (3CLpro) and the papain-like protease (PLpro). PLpro also has deubiquitinase (DUB) activity, removing ubiquitin (Ub) and Ub-like modifications from host proteins, disrupting the host immune response. 3CLpro is inhibited by many known cysteine protease inhibitors, whereas PLpro is a relatively unusual cysteine protease, being resistant to blockade by such inhibitors. A high-throughput screen of biased and unbiased libraries gave a low hit rate, identifying only CPI-169 and the positive control, GRL0617, as inhibitors with good potency (IC50 < 10 {micro}M). Analogues of both inhibitors were designed to develop structure-activity relationships; however, without a co-crystal structure of the CPI-169 series, we focused on GRL0617 as a starting point for structure-based drug design, obtaining several co-crystal structures to guide optimization. A series of novel 2-phenylthiophene-based non-covalent SARS-CoV-2 PLpro inhibitors were obtained, culminating in low nanomolar potency. The high potency and slow inhibitor off-rate were rationalized by newly identified ligand interactions with a "BL2 groove" that is distal from the active site cysteine. Trapping of the conformationally flexible BL2 loop by these inhibitors blocks binding of viral and host protein substrates; however, until now it has not been demonstrated that this mechanism can induce potent and efficacious antiviral activity. In this study, we report that novel PLpro inhibitors have excellent antiviral efficacy and potency against infectious SARS-CoV-2 replication in cell cultures. Together, our data provide structural insights into the design of potent PLpro inhibitors and the first validation that non-covalent inhibitors of SARS-CoV-2 PLpro can block infection of human cells with low micromolar potency.

microbiology↗