Infigratinib Is a Reversible Inhibitor and Mechanism-Based Inactivator of Cytochrome P450 3A4.

Infigratinib (INF) is a promising selective inhibitor of fibroblast growth factor receptors 1-3 that has recently been accorded both orphan drug designation and priority review status by the US Food and Drug Administration for the treatment of advanced cholangiocarcinoma. Its propensity to undergo bioactivation to electrophilic species was recently expounded upon. However, other than causing aberrant idiosyncratic toxicities, these reactive intermediates may elicit mechanism-based inactivation of cytochrome P450 enzymes. In this study, we investigated the interactions between INF and the most abundant hepatic CYP3A. Our findings revealed that, apart from being a potent noncompetitive reversible inhibitor of CYP3A4, INF inactivated CYP3A4 in a time-, concentration- and NADPH-dependent manner with inactivator concentration at half-maximum inactivation rate constant, maximum inactivation rate constant, and partition ratio of 4.17 µM, 0.068 minute-1, and 41, respectively, when rivaroxaban was employed as the probe substrate. Coincubation with testosterone (alternative CYP3A substrate) or ketoconazole (direct CYP3A inhibitor) attenuated the rate of inactivation, whereas the inclusion of glutathione and catalase did not confer such protection. The lack of enzyme activity recovery after dialysis for 4 hours and oxidation with potassium ferricyanide, coupled with the absence of the characteristic Soret peak signature collectively substantiated that inactivation of CYP3A4 by INF was not mediated by the formation of quasi-irreversible metabolite-intermediate complexes but rather through irreversible covalent adduction to the prosthetic heme and/or apoprotein. Finally, glutathione trapping and high-resolution mass spectrometry experimental results unraveled two plausible bioactivation mechanisms of INF arising from the generation of a p-benzoquinonediimine and epoxide reactive intermediate. SIGNIFICANCE STATEMENT: The potential of INF to cause MBI of CYP3A4 was unknown. This study reports the reversible noncompetitive inhibition and irreversible covalent MBI of CYP3A4 by INF and proposes two potential bioactivation pathways implicating p-benzoquinonediimine and epoxide reactive intermediates, following which a unique covalent docking methodology was harnessed to elucidate the structural and molecular determinants underscoring its inactivation. Findings from this study lay the groundwork for future investigation of clinically relevant drug-drug interactions between INF and concomitant substrates of CYP3A4.

Mechanism-Based Inactivation of Cytochrome P450 3A4 by Benzbromarone.

Benzbromarone (BBR), a potent uricosuric agent for the management of gout, is known to cause fatal fulminant hepatitis. Although the mechanism of BBR-induced idiosyncratic hepatotoxicity remains unelucidated, cytochrome P450 enzyme-mediated bioactivation of BBR to electrophilic reactive metabolites is commonly regarded as a key molecular initiating event. However, apart from causing aberrant toxicities, reactive metabolites may result in mechanism-based inactivation (MBI) of cytochrome P450. Here, we investigated and confirmed that BBR inactivated CYP3A4 in a time-, concentration-, and NADPH-dependent manner with K I, k inact, and partition ratio of 11.61 µM, 0.10 minutes-1, and 110, respectively. Coincubation with ketoconazole, a competitive inhibitor of CYP3A4, attenuated the MBI of CYP3A4 by BBR, whereas the presence of glutathione and catalase did not confer such protection. The lack of substantial recovery of enzyme activity postdialysis and after oxidation with potassium ferricyanide, combined with the absence of a Soret peak in spectral difference scans, implied that MBI of CYP3A4 by BBR did not occur through the formation of quasi-irreversible metabolite-intermediate complexes. Analysis of the reduced CO-difference spectrum revealed an ∼44% reduction in ferrous-CO binding and hinted that inactivation is mediated via irreversible covalent adduction to both the prosthetic heme moiety and the apoprotein. Finally, our in silico covalent docking analysis further suggested the modulation of substrate binding to CYP3A4 via the covalent adduction of epoxide-derived reactive intermediates of BBR to two key cysteine residues (Cys239 and Cys58) vicinal to the entrance of the orthosteric binding site. SIGNIFICANCE STATEMENT: Although the bioactivation of benzbromarone (BBR) to reactive metabolites has been well characterized, its potential to cause mechanism-based inactivation (MBI) of cytochrome P450 has not been fully investigated. This study reports the MBI of CYP3A4 by BBR via irreversible covalent adduction and develops a unique covalent docking methodology to predict the structural molecular determinants underpinning the inactivation for the first time. These findings lay the groundwork for future investigation of clinically relevant drug-drug interactions implicating BBR and mechanisms of BBR-induced idiosyncratic hepatotoxicity.

Gs- versus Golf-dependent functional selectivity mediated by the dopamine D1 receptor.

The two highly homologous subtypes of stimulatory G proteins Gαs (Gs) and Gαolf (Golf) display contrasting expression patterns in the brain. Golf is predominant in the striatum, while Gs is predominant in the cortex. Yet, little is known about their functional distinctions. The dopamine D1 receptor (D1R) couples to Gs/olf and is highly expressed in cortical and striatal areas, making it an important therapeutic target for neuropsychiatric disorders. Using novel drug screening methods that allow analysis of specific G-protein subtype coupling, we found that, relative to dopamine, dihydrexidine and N-propyl-apomorphine behave as full D1R agonists when coupled to Gs, but as partial D1R agonists when coupled to Golf. The Gs/Golf-dependent biased agonism by dihydrexidine was consistently observed at the levels of cellular signaling, neuronal function, and behavior. Our findings of Gs/Golf-dependent functional selectivity in D1R ligands open a new avenue for the treatment of cortex-specific or striatum-specific neuropsychiatric dysfunction.

Comparison of two different doses of intrathecal dexmedetomidine as adjuvant with isobaric ropivacaine in lower abdominal surgery.

BACKGROUND: To augment the subarachnoid block utility, the efficacy of newer molecules as an adjuvant is investigated constantly. Considering the favorable profile of dexmedetomidine, it could have a potential role as an adjuvant to ropivacaine. AIM: We evaluated the efficacy of two different doses of dexmedetomidine as an adjuvant to isobaric ropivacaine, intrathecally. METHODS: Ninety patients scheduled for lower abdominal surgery under spinal anesthesia were randomized into three groups to receive 2.5 ml of isobaric ropivacaine (0.75%, 7.5 mg/ml) added to 5 µg (10 µg/ml) or 10 µg (20 µg/ml) of dexmedetomidine or 0.5 ml of normal saline in group A, B or C, respectively. Block characteristics were compared as a primary outcome. STATISTICAL ANALYSIS: One-way analysis of variance test, Fisher's exact test/Chi-square test, whichever appropriate. A P < 0.05 was considered significant. RESULTS: Time to achieve desired block was least in group B and maximum in group C. The sensory-motor blockade remained significantly prolonged in group B compared to other groups. Hemodynamic parameters remained stable in all three groups. CONCLUSION: Among the investigated doses, dexmedetomidine augments the efficacy of intrathecal ropivacaine in a dose-dependent manner, without any untoward side effects.

Major intrinsic protein superfamily: channels with unique structural features and diverse selectivity filters.

Members of the superfamily of major intrinsic proteins (MIPs) facilitate water and solute permeability across cell membranes and are found in sources ranging from bacteria to humans. Aquaporin and aquaglyceroporin channels are the prominent members of the MIP superfamily. Experimental studies show that MIPs are involved in important physiological processes in mammals and plants. They are implicated in several human diseases and are considered to be attractive drug targets for a wide range of diseases such as cancer, brain edema, epilepsy, glaucoma, and congestive heart failure. Three-dimensional structures of MIP channels from diverse sources reveal that MIPs adopt a unique conserved hourglass helical fold consisting of six transmembrane helices (TM1-TM6) and two half-helices (LB and LE). Conserved NPA motifs near the center and the aromatic/arginine selectivity filter (Ar/R SF) toward the extracellular side constitute two narrow constriction regions within the channel. Structural knowledge combined with simulation studies have helped to investigate the role of these two constriction regions in the transport and selectivity of the solutes. With the availability of many genome sequences from diverse species, a large number of MIP genes have been identified. Homology models of 1500 MIP channels have been used to derive structure-based sequence alignment of TM1-TM6 helices and the two half-helices LB and LE. Thirteen residues are highly conserved in different transmembrane helices and half-helices. High group conservation of small and weakly polar residues is observed in 27 positions at the interface of two interacting helices. Thus, although the MIP sequences are diverse, the hourglass helical fold is maintained during evolution with the conservation of these 40 positions within the transmembrane region. We have proposed a generic structure-based numbering scheme for the MIP channels that will facilitate easier comparison of the MIP sequences. Analysis of Ar/R SF in all 1500 MIPs indicates the extent of diversity in the four residues that form this narrow region. Certain residues are completely avoided in the SF, even if they have the same chemical nature as that of the most frequently observed residues. For example, arginine is the most preferred residue in a specific position of Ar/R SF, whereas lysine is almost always avoided in any of the four positions. MIP channels with highly hydrophobic or hydrophilic Ar/R SF have been identified. Similarly, there are examples of MIP channels in which all four residues of Ar/R SF are bulky, thus almost occluding the pore. Many plant MIPs possess small residues at all SF positions, resulting in a larger pore diameter. A majority of MIP channels are yet to be functionally characterized, and their in vivo substrates are not yet identified. A complete understanding of the relationship between the nature of Ar/R SF and the solutes that are transported is required to exploit MIP channels as potential drug targets.