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

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.

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.

Regulation of Nuclear Receptors PXR and CAR by Small Molecules and Signal Crosstalk: Roles in Drug Metabolism and Beyond.

Pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are ligand-activated transcription factors that regulate the expression of drug metabolizing enzymes and drug transporters. Since their discoveries, they have been studied as important factors for regulating processes related to drug efficacy, drug toxicity, and drug-drug interactions. However, their vast ligand-binding profiles extend into additional spaces, such as endogenously produced chemicals, microbiome metabolites, dietary compounds, and environmental pollutants. Therefore, PXR and CAR can respond to an enormous abundance of stimuli, resulting in significant shifts in metabolic programs and physiologic homeostasis. Naturally, PXR and CAR have been implicated in various diseases related to homeostatic perturbations, such as inflammatory bowel disorders, diabetes, and certain cancers. Recent findings have injected the field with new signaling mechanisms and tools to dissect the complex PXR and CAR biology and have strengthened the potential for future PXR and CAR modulators in the clinic. Here, we describe the historical and ongoing importance of PXR and CAR in drug metabolism pathways and how this history has evolved into new mechanisms that regulate and are regulated by these xenobiotic receptors, with a specific focus on small molecule ligands. To effectively convey the impact of newly emerging research, we have arranged five diverse and representative key recent advances, four specific challenges, and four perspectives on future directions. SIGNIFICANCE STATEMENT: PXR and CAR are key transcription factors that regulate homeostatic detoxification of the liver and intestines. Diverse chemicals bind to these nuclear receptors, triggering their transcriptional tuning of the cellular metabolic response. This minireview revisits the importance of PXR and CAR in pharmaceutical drug responses and highlights recent results with implications beyond drug metabolism.

PROTAC-mediated activation, rather than degradation, of a nuclear receptor reveals complex ligand-receptor interaction network.

Proteolysis-targeting chimeras (PROTACs) are heterobifunctional molecules containing a ligand for a protein of interest linked to an E3 ubiquitin ligase ligand that induce protein degradation through E3 recruitment to the target protein. Small changes in PROTAC linkers can have drastic consequences, including loss of degradation activity, but the structural mechanisms governing such changes are unclear. To study this phenomenon, we screened PROTACs of diverse targeting modalities and identified dTAG-13 as an activator of the xenobiotic-sensing pregnane X receptor (PXR), which promiscuously binds various ligands. Characterization of dTAG-13 analogs and precursors revealed interplay between the PXR-binding moiety, linker, and E3 ligand that altered PXR activity without inducing degradation. A crystal structure of PXR ligand binding domain bound to a precursor ligand showed ligand-induced binding pocket distortions and a linker-punctured tunnel to the protein exterior at a region incompatible with E3 complex formation, highlighting the effects of linker environment on PROTAC activity.

First-in-Class Small Molecule Degrader of Pregnane X Receptor Enhances Chemotherapy Efficacy.

Pregnane X receptor (PXR) is a ligand-activated transcription factor that binds diverse compounds and upregulates drug metabolism machinery in response. PXR activation is detrimental to drug efficacy and safety because it reduces active drug concentrations and increases reactive metabolites, leading to toxicity and/or drug-drug interactions. Thus, effort must be expended in drug development pipelines to assess PXR activation by lead candidates and chemically modify agonists to reduce PXR liabilities while maintaining on-target potencies. Coadministration of drugs with PXR antagonists could prevent PXR-mediated metabolism events, but such compounds are rare and may themselves be converted to agonists by metabolic enzymes or PXR mutations. Here, we report the design, synthesis, optimization, and biological validation of proteolysis targeting chimeras that induce PXR degradation through E3 ubiquitin ligase recruitment. PXR degradation blocks agonist-induced gene expression and enhances anticancer effects of the chemotherapy paclitaxel, a known PXR agonist and substrate of downstream metabolic enzymes.

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.

Ligand flexibility and binding pocket malleability cooperate to allow selective PXR activation by analogs of a promiscuous nuclear receptor ligand.

The human nuclear receptor (NR) family of transcription factors contains 48 proteins that bind lipophilic molecules. Approved NR therapies have had immense success treating various diseases, but lack of selectivity has hindered efforts to therapeutically target the majority of NRs due to unpredictable off-target effects. The synthetic ligand T0901317 was originally discovered as a potent agonist of liver X receptors (LXRα/ß) but subsequently found to target additional NRs, with activation of pregnane X receptor (PXR) being as potent as that of LXRs. We previously showed that directed rigidity reduces PXR binding by T0901317 derivatives through unfavorable protein remodeling. Here, we use a similar approach to achieve selectivity for PXR over other T0901317-targeted NRs. One molecule, SJPYT-318, accomplishes selectivity by favorably utilizing PXR's flexible binding pocket and surprisingly binding in a new mode distinct from the parental T0901317. Our work provides a structure-guided framework to achieve NR selectivity from promiscuous compounds.

Bacteriological Assessment of the Indoor Air of Different Hospitals of Kathmandu District.

Nosocomial infection is the infection that has been caught in a hospital and is potentially caused by organisms that are not susceptible to antibiotics. Nosocomial infections are transmitted directly or indirectly through air and may cause different types of infections. This study was undertaken with an objective to determine the prevalence of nosocomial bacteria present in hospital indoor environment. A total of 16 air samples were taken from general wards and emergency wards of 8 different hospitals using an impactor air sampler in nutrient agar, mannitol salt agar, blood agar, cetrimide agar, and MacConkey agar. The bacteriological agents were isolated and identified by cultural characteristics, Gram staining, and biochemical tests, and their antibiotic susceptibility pattern was determined using CLSI Guideline, 2015. According to the European Union Guidelines to Good Manufacturing Practices, the hospitals were under C- and D-grade air quality. According to the European Commission, most of the hospitals were intermediately polluted. Out of 16 indoor air samples, 47.18% of Staphylococcus aureus and 1.82% Pseudomonas spp. were isolated. CoNS, Streptococcus spp., Micrococcus spp., and Bacillus spp. and Gram-negative bacteria E.coli and Proteus spp. were identified. The bacterial load was found to be high in the emergency ward (55.8%) in comparison to that in the general ward (44.2%). There is statistically no significant difference between bacterial load and 2 wards (general and emergency) of different hospitals and among different hospitals. The most effective antibiotic against S. aureus was gentamicin (81.81%) and ofloxacin (81.81%). Among the antibiotics used for Pseudomonas spp., ceftriaxone (83.3%) and ofloxacin (83.3%) were effective. High prevalence of S. aureus and Gram-negative bacteria was found in this study; it is therefore important to monitor air quality regularly at different hospitals to prevent HAI.