Comparison of the CYP3A selective inhibitors CYP3cide, clobetasol, and azamulin for their potential to distinguish CYP3A7 activity in the presence of CYP3A4/5.

The CYP3A7 enzyme accounts for ~50% of the total CYP content in fetal and neonatal livers and is the predominant CYP involved in neonatal xenobiotic metabolism. Additionally, it is a key player in healthy birth outcomes through the oxidation of dehydroepiandrosterone (DHEA) and DHEA-S (sulfate). The amount of the other hepatic CYP3A isoforms, CYP3A4 and CYP3A5, expressed in neonates is low, but highly variable, and therefore the activity of individual CYP3A isoforms is difficult to differentiate due to their functional similarities. Consequently, a better understanding of the contribution of CYP3A7 to drug metabolism is essential to identify the risk drugs may pose to neonates and developing infants. To distinguish CYP3A7 activity from CYP3A4/5, we sought to further characterize the selectivity of the specific CYP3A inhibitors CYP3cide, clobetasol, and azamulin. We utilized three substrate probes, dibenzylfluorescein, luciferin-PPXE, and midazolam, to determine the IC50 and metabolism-dependent inhibition (MDI) properties of the CYP3A inhibitors. Probe selection had a significant effect on the IC50 values and CYP inactivation across all inhibitory compounds and enzymes. CYP3cide and azamulin were both identified as MDIs and were most specific for CYP3A4. Contrary to previous reports, we found that CP was not an MDI of CYP3A5, but was more selective for CYP3A5 over CYP3A4/7. We further investigated CYP3cide and CP's ability to differentiate CYP3A7 activity in an equal mixture of recombinant CYP3A4, CYP3A5, and CYP3A7 and our results provide confidence of CYP3cide's and CP's ability to distinguish CYP3A7 activity in the presence of the other CYP3A isoforms. Significance Statement These findings provide valuable insight regarding in vitro testing conditions to investigate the metabolism of new drug candidates and help determine drug safety in neonates. The results presented here also clearly demonstrate the effect probe selection may have on CYP3A P450 inhibition studies.

Consideration of Nevirapine Analogs To Reduce Metabolically Linked Hepatotoxicity: A Cautionary Tale of the Deuteration Approach.

Idiosyncratic drug reactions (IDRs) in their most deleterious form can lead to serious medical complications and potentially fatal events. Nevirapine (NVP), still widely used in developing countries for combinatorial antiretroviral and prophylactic therapies against HIV infection, represents a prototypical example of IDRs causing severe skin rashes and hepatotoxicity. Complex metabolic pathways accompanied by production of multiple reactive metabolites often complicate our understanding of IDR's origin. While assessment of NVP analogs has helped characterize the pathways involved in IDRs for NVP, which are largely driven by metabolism at the 12-methyl position, it has yet to be investigated if some of these analogs could be valuable replacement drugs with reduced reactive metabolite properties and drug-drug interaction (DDI) risks. Here, we evaluated a set of eight NVP analogs, including the deuterated 12-d3-NVP and two NVP metabolites, for their efficacy and inhibitory potencies against HIV reverse transcriptase (HIV-RT). A subset of three analogs, demonstrating >85% inhibition for HIV-RT, was further assessed for their hepatic CYP induction-driven DDI risks. This led to a closer investigation of the inactivation properties of 12-d3-NVP for hepatic CYP3A4 and a comparison of its propensity in generating reactive metabolite species. The metabolic shift triggered with 12-d3-NVP, increasing formation of the 2-hydroxy and glutathione metabolites, emphasized the importance of the dynamic balance between induction and metabolism-dependent inactivation of CYP3A4 and its impact on clearance of NVP during treatment. Unfortunately, the strategy of incorporating deuterium to reduce NVP metabolism and production of the electrophile species elicited opposite results, illustrating the great challenges involved in tackling IDRs through deuteration.

Human cytochrome P450 3A7 binding four copies of its native substrate dehydroepiandrosterone 3-sulfate.

Human fetal cytochrome P450 3A7 (CYP3A7) is involved in both xenobiotic metabolism and the estriol biosynthetic pathway. Although much is understood about cytochrome P450 3A4 and its role in adult drug metabolism, CYP3A7 is poorly characterized in terms of its interactions with both categories of substrates. Herein, a crystallizable mutated form of CYP3A7 was saturated with its primary endogenous substrate dehydroepiandrosterone 3-sulfate (DHEA-S) to yield a 2.6 Å X-ray structure revealing the unexpected capacity to simultaneously bind four copies of DHEA-S. Two DHEA-S molecules are located in the active site proper, one in a ligand access channel, and one on the hydrophobic F'-G' surface normally embedded in the membrane. While neither DHEA-S binding nor metabolism exhibit cooperative kinetics, the current structure is consistent with cooperativity common to CYP3A enzymes. Overall, this information suggests that mechanism(s) of CYP3A7 interactions with steroidal substrates are complex.

Per- and polyfluoroalkyl substances (PFAS) inhibit cytochrome P450 CYP3A7 through direct coordination to the heme iron and water displacement.

Per- and polyfluoroalkyl substances (PFAS) are a chemical class of highly stable, fluorinated compounds popular for use in a variety of consumer products. PFAS environmental persistence in drinking water contributes to acute exposure in humans and subsequent bioaccumulation of the compounds in the liver and lung tissue. Prenatal PFAS exposure has been associated with lowered birth weight, premature birth, and developmental defects including cranio-facial abnormalities. The cytochrome P450 enzyme CYP3A7 is responsible for facilitating a variety of reactions essential for proper fetal development in humans. In addition to drug metabolism, CYP3A7 is responsible for metabolizing endogenous ligands in the developing human liver, including the steroid precursor dehydroepiandrosterone 3-sulfate (DHEA-S), essential for estriol synthesis during pregnancy, along with the morphogen all-trans-retinoic acid (atRA). Interference with estriol synthesis during pregnancy, as well as atRA clearance, is known to result in similar effects associated with prenatal PFAS exposure including lowered birth weight, premature birth, and developmental defects. We hypothesized that PFAS compounds bind to the CYP3A7 enzyme resulting in its inhibition. We implemented a series of binding studies using spectral characterization of six PFAS compounds (PFOA, PFOS, GenX, PFNA, PFNS, and PFHxS), and evaluated their interactions with recombinant CYP3A7. In addition, we screened PFAS for their ability to inhibit CYP3A7 oxidative activity using dibenzylfluorescein, a fluorescent probe, and DHEA-S, an endogenous substrate of CYP3A7. Our data demonstrate that of the six PFAS tested, PFOA, PFOS, PFNA, and PFHxS bind to and inhibit CYP3A7.

Identification of Aloe-derived natural products as prospective lead scaffolds for SARS-CoV-2 main protease (Mpro) inhibitors.

In the past two years, the COVID-19 pandemic has caused over 5 million deaths and 250 million infections worldwide. Despite successful vaccination efforts and emergency approval of small molecule therapies, a diverse range of antivirals is still needed to combat the inevitable resistance that will arise from new SARS-CoV-2 variants. The main protease of SARS-CoV-2 (Mpro) is an attractive drug target due to the clinical success of protease inhibitors against other viruses, such as HIV and HCV. However, in order to combat resistance, various chemical scaffolds need to be identified that have the potential to be developed into potent inhibitors. To this end, we screened a high-content protease inhibitor library against Mproin vitro, in order to identify structurally diverse compounds that could be further developed into antiviral leads. Our high-content screening efforts retrieved 27 hits each with > 50% inhibition in our Mpro FRET assay. Of these, four of the top inhibitor compounds were chosen for follow-up due to their potency and drugability (Lipinski's rules of five criteria): anacardic acid, aloesin, aloeresin D, and TCID. Further analysis via dose response curves revealed IC50 values of 6.8 µM, 38.9 µM, 125.3 µM, and 138.0 µM for each compound, respectively. Molecular docking studies demonstrated that the four inhibitors bound at the catalytic active site of Mpro with varying binding energies (-7.5 to -5.6 kcal/mol). Furthermore, Mpro FRET assay kinetic studies demonstrated that Mpro catalysis is better represented by a sigmoidal Hill model than the standard Michaelis-Menten hyperbola, indicating substantial cooperativity of the active enzyme dimer. This result suggests that the dimerization interface could be an attractive target for allosteric inhibitors. In conclusion, we identified two closely-related natural product compounds from the Aloe plant (aloesin and aloeresin D) that may serve as novel scaffolds for Mpro inhibitor design and additionally confirmed the strongly cooperative kinetics of Mpro proteolysis. These results further advance our knowledge of structure-function relationships in Mpro and offer new molecular scaffolds for inhibitor design.

Pseudomonas aeruginosa cytochrome P450 CYP168A1 is a fatty acid hydroxylase that metabolizes arachidonic acid to the vasodilator 19-HETE.

Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen that is highly prevalent in individuals with cystic fibrosis (CF). A major problem in treating CF patients infected with P. aeruginosa is the development of antibiotic resistance. Therefore, the identification of novel P. aeruginosa antibiotic drug targets is of the utmost urgency. The genome of P. aeruginosa contains four putative cytochrome P450 enzymes (CYPs) of unknown function that have never before been characterized. Analogous to some of the CYPs from Mycobacterium tuberculosis, these P. aeruginosa CYPs may be important for growth and colonization of CF patients' lungs. In this study, we cloned, expressed, and characterized CYP168A1 from P. aeruginosa and identified it as a subterminal fatty acid hydroxylase. Spectral binding data and computational modeling of substrates and inhibitors suggest that CYP168A1 has a large, expansive active site and preferentially binds long chain fatty acids and large hydrophobic inhibitors. Furthermore, metabolic experiments confirm that the enzyme is capable of hydroxylating arachidonic acid, an important inflammatory signaling molecule present in abundance in the CF lung, to 19-hydroxyeicosatetraenoic acid (19-HETE; Km = 41 µM, Vmax = 220 pmol/min/nmol P450), a potent vasodilator, which may play a role in the pathogen's ability to colonize the lung. Additionally, we found that the in vitro metabolism of arachidonic acid is subject to substrate inhibition and is also inhibited by the presence of the antifungal agent ketoconazole. This study identifies a new metabolic pathway in this important human pathogen that may be of utility in treating P. aeruginosa infections.

Characterization of fluorescent probe substrates to develop an efficient high-throughput assay for neonatal hepatic CYP3A7 inhibition screening.

CYP3A7 is a member of the cytochrome P450 (CYP) 3A enzyme sub-family that is expressed in the fetus and neonate. In addition to its role metabolizing retinoic acid and the endogenous steroid dehydroepiandrosterone sulfate (DHEA-S), it also has a critical function in drug metabolism and disposition during the first few weeks of life. Despite this, it is generally ignored in the preclinical testing of new drug candidates. This increases the risk for drug-drug interactions (DDI) and toxicities occurring in the neonate. Therefore, screening drug candidates for CYP3A7 inhibition is essential to identify chemical entities with potential toxicity risks for neonates. Currently, there is no efficient high-throughput screening (HTS) assay to assess CYP3A7 inhibition. Here, we report our testing of various fluorescent probes to assess CYP3A7 activity in a high-throughput manner. We determined that the fluorescent compound dibenzylfluorescein (DBF) is superior to other compounds in meeting the criteria considered for an efficient HTS assay. Furthermore, a preliminary screen of an HIV/HCV antiviral drug mini-library demonstrated the utility of DBF in a HTS assay system. We anticipate that this tool will be of great benefit in screening drugs that may be used in the neonatal population in the future.

Inhibition of CYP3A7 DHEA-S Oxidation by Lopinavir and Ritonavir: An Alternative Mechanism for Adrenal Impairment in HIV Antiretroviral-Treated Neonates.

Prophylactic antiretroviral therapy (ART) in HIV infected pregnant mothers and their newborns can dramatically reduce mother-to-child viral transmission and seroconversion in the neonate. The ritonavir-boosted lopinavir regimen, known as Kaletra, has been associated with premature birth and transient adrenal insufficiency in newborns, accompanied by increases in plasma dehydroepiandrosterone 3-sulfate (DHEA-S). In the fetus and neonates, cytochrome P450 CYP3A7 is responsible for the metabolism of DHEA-S into 16α-hydroxy DHEA-S, which plays a critical role in growth and development. In order to determine if CYP3A7 inhibition could lead to the adverse outcomes associated with Kaletra therapy, we conducted in vitro metabolic studies to determine the extent and mechanism of CYP3A7 inhibition by both ritonavir and lopinavir and the relative intrinsic clearance of lopinavir with and without ritonavir in both neonatal and adult human liver microsomes (HLMs). We identified ritonavir as a potent inhibitor of CYP3A7 oxidation of DHEA-S (IC50 = 0.0514 µM), while lopinavir is a much weaker inhibitor (IC50 = 5.88 µM). Furthermore, ritonavir is a time-dependent inhibitor of CYP3A7 with a KI of 0.392 µM and a kinact of 0.119 min-1, illustrating the potential for CYP3A mediated drug-drug interactions with Kaletra. The clearance rate of lopinavir in neonatal HLMs was much slower and comparable to the rate observed in adult HLMs in the presence of ritonavir, suggesting that the addition of ritonavir in the cocktail therapy may not be necessary to maintain effective concentrations of lopinavir in neonates. Our results suggest that several of the observed adverse outcomes of Kaletra therapy may be due to the direct inhibition of CYP3A7 by ritonavir and that the necessity for the inclusion of this drug in the therapy may be obviated by the lower rate of lopinavir clearance in the neonatal liver. These results may lead to a reconsideration of the use of ritonavir in neonatal antiretroviral therapy.

Modulation of O-GlcNAc Levels in the Liver Impacts Acetaminophen-Induced Liver Injury by Affecting Protein Adduct Formation and Glutathione Synthesis.

Overdose of acetaminophen (APAP) results in acute liver failure. We have investigated the role of a posttranslational modification of proteins called O-GlcNAcylation, where the O-GlcNAc transferase (OGT) adds and O-GlcNAcase (OGA) removes a single ß-D-N-acetylglucosamine (O-GlcNAc) moiety, in the pathogenesis of APAP-induced liver injury. Hepatocyte-specific OGT knockout mice (OGT KO), which have reduced O-GlcNAcylation, and wild-type (WT) controls were treated with 300 mg/kg APAP and the development of injury was studied over a time course from 0 to 24 h. OGT KO mice developed significantly lower liver injury as compared with WT mice. Hepatic CYP2E1 activity and glutathione (GSH) depletion following APAP treatment were not different between WT and OGT KO mice. However, replenishment of GSH and induction of GSH biosynthesis genes were significantly faster in the OGT KO mice. Next, male C57BL/6 J mice were treated Thiamet-G (TMG), a specific inhibitor of OGA to induce O-GlcNAcylation, 1.5 h after APAP administration and the development of liver injury was studied over a time course of 0-24 h. TMG-treated mice exhibited significantly higher APAP-induced liver injury. Treatment with TMG did not affect hepatic CYP2E1 levels, GSH depletion, APAP-protein adducts, and APAP-induced mitochondrial damage. However, GSH replenishment and GSH biosynthesis genes were lower in TMG-treated mice after APAP overdose. Taken together, these data indicate that induction in cellular O-GlcNAcylation exacerbates APAP-induced liver injury via dysregulation of hepatic GSH replenishment response.

Variability in Potency Among Commercial Preparations of Berberine.

Berberine is an isoquinoline alkaloid plant extract that is widely available as a dietary supplement in the United States and has demonstrated efficacy in the treatment of type 2 diabetes mellitus and dyslipidemia. Because of its increased use and purported pharmacological properties, potential variations in product quality could pose a barrier to berberine's safety and effectiveness in clinical practice. Thus, this study evaluated the potency of dietary supplements containing berberine available in the U.S. commercial market. Fifteen unique dietary supplements containing berberine were purchased through U.S. dietary supplement vendors. For each product, berberine was extracted from 3 unique capsules and analyzed by ultra-high-performance liquid chromatography tandem mass spectrometry. Percentage content based on the product label claim was determined for each product. The average berberine content across the products was found to be 75% ± 25% of the product label claim, with product potency ranging from 33% to 100%. Nine of the 15 tested products (60%) failed to meet the potency standards of 90% to 110% of labeled content claim, as commonly required of pharmaceutical preparations by the U.S. Pharmacopeial Convention. Evaluation of the relationship between product cost and the measured potency failed to demonstrate an association between quality and cost. Variability in product quality may significantly contribute to inconsistencies in the safety and effectiveness of berberine. In addition, the quality of the berberine product cannot be inferred from its cost.

Digging Deeper into CYP3A Testosterone Metabolism: Kinetic, Regioselectivity, and Stereoselectivity Differences between CYP3A4/5 and CYP3A7.

The metabolism of testosterone to 6ß-hydroxytestosterone (6ß-OH-T) is a commonly used assay to evaluate human CYP3A enzyme activities. However, previous reports have indicated that CYP3A7 also produces 2α-hydroxytestosterone (2α-OH-T) and that a 2α-OH-T/6ß-OH-T ratio may be a unique endogenous biomarker of the activity of the enzyme. Until now, the full metabolite and kinetic profile for testosterone hydroxylation by CYP3A7 has not been fully examined. To this end, we performed a complete kinetic analysis of the 6ß-OH-T, 2α-OH-T, and 2ß-hydroxytestosterone metabolites for recombinant Supersome CYP3A4, CYP3A5, and CYP3A7 enzymes and monitored metabolism in fetal and adult human liver microsomes for comparison. In general, a decrease in the velocity of the reaction was observed between CYP3A4 and the two other enzymes, with CYP3A7 showing the lowest metabolic capacity. Interestingly, we found that the 2α-OH-T/6ß-OH-T ratio varied with substrate concentration when testosterone was incubated with CYP3A7, suggesting that this ratio would likely not function well as a biomarker for CYP3A7 activity. In silico docking studies revealed at least two different binding modes for testosterone between CYP3A4 and CYP3A7. In CYP3A4, the most energetically favorable docking mode places testosterone in a position with the methyl groups directed toward the heme iron, which is more favorable for oxidation at C6ß, whereas for CYP3A7 the testosterone methyl groups are positioned away from the heme, which is more favorable for an oxidation event at C2α In conclusion, our data indicate an alternative binding mode for testosterone in CYP3A7 that favors the 2α-hydroxylation, suggesting significant structural differences in its active site compared with CYP3A4/5.

Role of protein-protein interactions in cytochrome P450-mediated drug metabolism and toxicity.

Through their unique oxidative chemistry, cytochrome P450 monooxygenases (CYPs) catalyze the elimination of most drugs and toxins from the human body. Protein-protein interactions play a critical role in this process. Historically, the study of CYP-protein interactions has focused on their electron transfer partners and allosteric mediators, cytochrome P450 reductase and cytochrome b5. However, CYPs can bind other proteins that also affect CYP function. Some examples include the progesterone receptor membrane component 1, damage resistance protein 1, human and bovine serum albumin, and intestinal fatty acid binding protein, in addition to other CYP isoforms. Furthermore, disruption of these interactions can lead to altered paths of metabolism and the production of toxic metabolites. In this review, we summarize the available evidence for CYP protein-protein interactions from the literature and offer a discussion of the potential impact of future studies aimed at characterizing noncanonical protein-protein interactions with CYP enzymes.

Cytochrome P450 Enzyme Metabolites in Lead Discovery and Development.

The cytochrome P450 (CYP) enzymes are a versatile superfamily of heme-containing monooxygenases, perhaps best known for their role in the oxidation of xenobiotic compounds. However, due to their unique oxidative chemistry, CYPs are also important in natural product drug discovery and in the generation of active metabolites with unique therapeutic properties. New tools for the analysis and production of CYP metabolites, including microscale analytical technologies and combinatorial biosynthesis, are providing medicinal chemists with the opportunity to use CYPs as a novel platform for lead discovery and development. In this review, we will highlight some of the recent examples of drug leads identified from CYP metabolites and the exciting possibilities of using CYPs as catalysts for future drug discovery.