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Miller, Darcie J.

Publications and source records attributed to Miller, Darcie J..

15 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Chemical manipulation of an activation/inhibition switch in the nuclear receptor PXR

Nuclear receptors are ligand-activated transcription factors that can often be useful drug targets. Unfortunately, ligand promiscuity leads to two-thirds of receptors remaining clinically untargeted. PXR is a nuclear receptor that can be activated by diverse compounds to elevate metabolism, negatively impacting drug efficacy and safety. This presents a barrier to drug development because compounds designed to target other proteins must avoid PXR activation while retaining potency for the desired target. This problem could be avoided by using PXR antagonists, but these compounds are rare, and their molecular mechanisms remain unknown. Here, we report structurally related PXR-selective agonists and antagonists and their corresponding co-crystal structures to describe mechanisms of antagonism and selectivity. Structural and computational approaches show that antagonists induce PXR conformational changes incompatible with transcriptional coactivator recruitment. These results guide the design of compounds with predictable agonist/antagonist activities and bolster efforts to generate antagonists to prevent PXR activation interfering with other drugs.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Selective CK1α degraders exert antiproliferative activity against a broad range of human cancer cell lines

Molecular-glue degraders are small molecules that induce a specific interaction between an E3 ligase and a target protein, resulting in the target proteolysis. The discovery of molecular glue degraders currently relies mostly on screening approaches. Here, we describe screening of a library of cereblon (CRBN) ligands against a panel of patient-derived cancer cell lines, leading to the discovery of SJ7095, a potent degrader of CK1α, IKZF1 and IKZF3 proteins. Through a structure-informed exploration of structure activity relationship (SAR) around this small molecule we develop SJ3149, a selective and potent degrader of CK1α protein in vitro and in vivo. The structure of SJ3149 co-crystalized in complex with CK1α + CRBN + DDB1 provides a rationale for the improved degradation properties of this compound. In a panel of 115 cancer cell lines SJ3149 displays a broad antiproliferative activity profile, which shows statistically significant correlation with MDM2 inhibitor Nutlin-3a. These findings suggest potential utility of selective CK1α degraders for treatment of hematological cancers and solid tumors.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

A bromodomain-independent mechanism of gene regulation by the BET inhibitor JQ1: direct activation of nuclear receptor PXR

Abstract Bromodomain and extraterminal (BET) proteins are extensively studied in multiple pathologies, including cancer. BET proteins modulate transcription of various genes, including those synonymous with cancer, such as MYC. Thus, BET inhibitors are a major area of drug development efforts. (+)-JQ1 (JQ1) is the prototype inhibitor and is a common tool to probe BET functions. While showing therapeutic promise, JQ1 is not clinically usable, partly due to metabolic instability. Here, we show that JQ1 and the BET-inactive (−)-JQ1 are agonists of pregnane X receptor (PXR), a nuclear receptor that transcriptionally regulates genes encoding drug-metabolizing enzymes such as CYP3A4, which was previously shown to oxidize JQ1. A PXR-JQ1 co-crystal structure identified JQ1′s tert-butyl moiety as a PXR anchor and explains binding by (−)-JQ1. Analogs differing at the tert-butyl lost PXR binding, validating our structural findings. Evaluation in liver cell models revealed both PXR-dependent and PXR-independent modulation of CYP3A4 expression by BET inhibitors. We have characterized a non-BET JQ1 target, a mechanism of physiological JQ1 instability, a biological function of (−)-JQ1, and BET-dependent transcriptional regulation of drug metabolism genes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structure-guided approach to modulate small molecule binding to a promiscuous ligand-activated protein

Ligand-binding promiscuity in detoxification systems protects the body from toxicological harm but is a roadblock to drug development due to the difficulty in optimizing small molecules to both retain target potency and avoid metabolic events. Immense effort is invested in evaluating metabolism of molecules to develop safer, more effective treatments, but engineering specificity into or out of promiscuous proteins and their ligands is a challenging task. To better understand the promiscuous nature of detoxification networks, we have used X-ray crystallography to characterize a structural feature of pregnane X receptor (PXR), a nuclear receptor that is activated by diverse molecules (with different structures and sizes) to up-regulate transcription of drug metabolism genes. We found that large ligands expand PXR’s ligand-binding pocket, and the ligand-induced expansion occurs through a specific unfavorable compound-protein clash that likely contributes to reduced binding affinity. Removing the clash by compound modification resulted in more favorable binding modes with significantly enhanced binding affinity. We then engineered the unfavorable ligand-protein clash into a potent, small PXR ligand, resulting in marked reduction in PXR binding and activation. Structural analysis showed that PXR is remodeled, and the modified ligands reposition in the binding pocket to avoid clashes, but the conformational changes result in less favorable binding modes. Thus, ligand-induced binding pocket expansion increases ligand-binding potential of PXR but is an unfavorable event; therefore, drug candidates can be engineered to expand PXR’s ligand-binding pocket and reduce their safety liability due to PXR binding.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Structural basis for substrate recognition and chemical inhibition of oncogenic MAGE ubiquitin ligases

Testis-restricted melanoma antigen (MAGE) proteins are frequently hijacked in cancer and play a critical role in tumorigenesis. MAGEs assemble with E3 ubiquitin ligases and function as substrate adaptors that direct the ubiquitination of novel targets, including key tumor suppressors. However, how MAGEs recognize their targets is unknown and has impeded the development of MAGE-directed therapeutics. Here, we report the structural basis for substrate recognition by MAGE ubiquitin ligases. Biochemical analysis of the degron motif recognized by MAGE-A11 and the crystal structure of MAGE-A11 bound to the PCF11 substrate uncovered a conserved substrate binding cleft (SBC) in MAGEs. Mutation of the SBC disrupted substrate recognition by MAGEs and blocked MAGE-A11 oncogenic activity. A chemical screen for inhibitors of MAGE-A11:substrate interaction identified 4-Aminoquinolines as potent inhibitors of MAGE-A11 that show selective cytotoxicity. These findings provide important insights into the large family of MAGE ubiquitin ligases and identify approaches for developing cancer-specific therapeutics.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2019

Ureadepsipeptides as ClpP Activators

Acyldepsipeptides are a unique class of antibiotics that act via allosterically dysregulated activation of the bacterial caseinolytic protease (ClpP). The ability of ClpP activators to kill nongrowing bacteria represents a new opportunity to combat deep-seated biofilm infections. However, the acyldepsipeptide scaffold is subject to rapid metabolism. In this study, we explore alteration of the potentially metabolically reactive α,β unsaturated acyl chain. Through targeted synthesis, a new class of phenyl urea substituted depsipeptide ClpP activators with improved metabolic stability is described. The ureadepsipeptides are potent activators of Staphylococcus aureus ClpP and show activity against Gram-positive bacteria, including S. aureus biofilms. These studies demonstrate that a phenyl urea motif can successfully mimic the double bond, maintaining potency equivalent to acyldepsipeptides but with decreased metabolic liability. Although removal of the double bond from acyldepsipeptides generally has a significant negative impact on potency, structural studies revealed that the phenyl ureadepsipeptides can retain potency through the formation of a third hydrogen bond between the urea and the key Tyr63 residue in the ClpP activation domain. Ureadepsipeptides represent a new class of ClpP activators with improved drug-like properties, potent antibacterial activity, and the tractability to be further optimized.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2019

Protein engineering of a ubiquitin-variant inhibitor of APC/C identifies a cryptic K48 ubiquitin chain binding site

Ubiquitin (Ub)-mediated proteolysis is a fundamental mechanism used by eukaryotic cells to maintain homeostasis and protein quality, and to control timing in biological processes. Two essential aspects of Ub regulation are conjugation through E1-E2-E3 enzymatic cascades and recognition by Ub-binding domains. An emerging theme in the Ub field is that these 2 properties are often amalgamated in conjugation enzymes. In addition to covalent thioester linkage to Ub’s C terminus for Ub transfer reactions, conjugation enzymes often bind noncovalently and weakly to Ub at “exosites.” However, identification of such sites is typically empirical and particularly challenging in large molecular machines. In this work, studying the 1.2-MDa E3 ligase anaphase-promoting complex/cyclosome (APC/C), which controls cell division and many aspects of neurobiology, we discover a method for identifying unexpected Ub-binding sites. Using a panel of Ub variants (UbVs), we identify a protein-based inhibitor that blocks Ub ligation to APC/C substrates in vitro and ex vivo. Biochemistry, NMR, and cryo-electron microscopy (cryo-EM) structurally define the UbV interaction, explain its inhibitory activity through binding the surface on the APC2 subunit that recruits the E2 enzyme UBE2C, and ultimately reveal that this APC2 surface is also a Ub-binding exosite with preference for K48-linked chains. The results provide a tool for probing APC/C activity, have implications for the coordination of K48-linked Ub chain binding by APC/C with the multistep process of substrate polyubiquitylation, and demonstrate the power of UbV technology for identifying cryptic Ub-binding sites within large multiprotein complexes.

59 BASIC BIOLOGICAL SCIENCES↗