Structure-based amelioration of PXR transactivation in a novel series of macrocyclic allosteric inhibitors of HIV-1 integrase

Previous studies have identified a series of imidazo[1,2-a]pyridine (IZP) derivatives as potent allosteric inhibitors of HIV-1 integrase (ALLINIs) and virus infection in cell culture. However, IZPs were also found to be relatively potent activators of the pregnane-X receptor (PXR), raising the specter of induction of CYP-mediated drug disposition pathways. In an attempt to modify PXR activity without affecting anti-HIV-1 activity, rational structure-based design and modeling approaches were used. An X-ray cocrystal structure of (S,S)-1 in the PXR ligand binding domain (LBD) allowed an examination of the potential of rational structural modifications designed to abrogate PXR. The introduction of bulky basic amines at the C-8 position provided macrocyclic IZP derivatives that displayed potent HIV-1 inhibitory activity in cell culture with no detectable PXR transactivation at the highest concentration tested.     

Influence of MDR1 C1236T polymorphism on lopinavir plasma concentration and virological response in HIV-1-infected children

Background

Variability in MDR1 and PXR has been associated with differences in drug plasma levels and response to antiretroviral therapy. We investigated whether polymorphisms in MDR1 (T-129C, C1236T and C3435T) and PXR (C63396T) affect lopinavir plasma concentration and the virological or immunological response to HAART in HIV-1-infected children.

Methods

Genotypes were identified in 100 blood donors and 38 HIV-1-infected children. All children received HAART with lopinavir boosted with ritonavir (LPV/r) at the time of LPV plasma level quantification, before (C_trough) and between 1 and 2 h after (C_post-dose) the administration of the next dose of drug. CD4⁺ T-cell counts and plasma viral load were analyzed before and after the initiation of LPV/r.

Results

MDR1 1236T, MDR1 3435T and PXR 63396T alleles showed a frequency of ~ 50% while the MDR1 -129C allele only reached 5%. Children heterozygotes 1236CT showed a significantly lower LPV C_post-dose than homozygotes 1236TT (median C_post-dose = 3.04 μg/ml and 6.50 μg/ml, respectively; p = 0.016). Children heterozygotes 1236CT also had a lower decrease of viral load after 36 weeks of LPV/r exposure compared with homozygotes 1236CC (median viral load changes = − 0.50 log₁₀ copies/ml and − 2.08 log₁₀ copies/ml, respectively; p = 0.047). No effect on the immunological response was observed for polymorphisms of MDR1 or PXR.

Conclusions

Our results suggest that the MDR1 C1236T SNP significantly reduces LPV plasma concentration affecting the virological response to HAART. Heterozygotes 1236CT might have an altered level of P-gp expression/activity in enterocytes and CD4⁺ T lymphocytes that limits the absorption of LPV leading to an impaired virological suppression.     

Prognostic molecular markers in pediatric liver disease – Are there any?

Pediatric liver disease (PLD) is a major cause of severe morbidity and prolonged hospitalizations in children. Stratifying patients in terms of prognosis remains challenging. The limited knowledge about molecular mechanisms causing and accompanying PLD remains the main obstacle in a search for reliable prognostic biomarkers. A systematic search of MEDLINE via PubMed and Embase via OVID was conducted on studies published between August 2007 and August 2017. Molecular markers with a prognostic potential in terms of survival, need for liver transplantation or disease progression/regression were selected. In general, identified studies were single center smaller case-control studies or case series with a low level of evidence and a high risk of bias. Only 23 studies comprising 898 patients could be included, mostly focusing on biliary atresia, non-alcoholic fatty liver disease, viral hepatitis, and LT; and markers related to morphogenesis and fibrosis. Furthermore, molecular markers in metabolic pathways and inflammation shown to be relevant, however requiring further validation. Hence, further biological and clinical studies are needed to gain greater molecular insight into PLD.     

Design and synthesis of lactam–thiophene carboxylic acids as potent hepatitis C virus polymerase inhibitors

Herein we report the successful incorporation of a lactam as an amide replacement in the design of hepatitis C virus NS5B Site II thiophene carboxylic acid inhibitors. Optimizing potency in a replicon assay and minimizing potential risk for CYP3A4 induction led to the discovery of inhibitor 22a. This lead compound has a favorable pharmacokinetic profile in rats and dogs.     

Natural products as modulators of the nuclear receptors and metabolic sensors LXR,FXR and RXR

Nuclear receptors (NRs) represent attractive targets for the treatment of metabolic syndrome-related diseases. In addition, natural products are an interesting pool of potential ligands since they have been refined under evolutionary pressure to interact with proteins or other biological targets.This review aims to briefly summarize current basic knowledge regarding the liver X (LXR) and farnesoid X receptors (FXR) that form permissive heterodimers with retinoid X receptors (RXR). Natural product-based ligands for these receptors are summarized and the potential of LXR, FXR and RXR as targets in precision medicine is discussed.     

Ectopic expression of MHC class II genes (RT1.B(I) beta/alpha) in rat hepatocytes in vivo and in culture can be elicited by treatment with the pregnane X receptor agonists pregnenolone 16 alpha-carbonitrile and dexamethasone

The synthetic steroid, pregnenolone-16-alpha-carbonitrile (PCN), has served for decades as a probe for a postulated series of hepatic defenses activated under situations of environmental "stress". PCN, an antiglucocorticoid, and also such glucocorticoids as dexamethasone (Dex) appear to stimulate hepatic metabolism and elimination of xenobiotics by binding to the nuclear pregnane X receptor (PXR) which then interacts with a distinct DNA response element associated with induction of cytochrome P450 3A genes. To explore the full domain of genes controlled by PCN/PXR, we used differential display to detect rat liver mRNA species selectively induced by PCN or by Dex. Sequence analysis identified one of many PCN induced cDNA fragments as RT1.B(I)beta, a member of the major histocompatability class II (MHC) gene family usually found only in antigen presenting cells. Northern blot analysis of RNA from rat liver or from cultured hepatocytes confirmed that amounts of RT1.B(I)beta mRNA and also of its companion gene, RT1.B(I)alpha mRNA, became readily detectable within 3-6 hours following treatment with PCN or Dex, whereas no induction was observed in spleen RNA. Induction by PCN of RT1.B(I)beta immunoreactive protein was localized to the hepatocytes as judged by immunofluorescence. We conclude that ectopic expression of MHC II genes, an unprecedented effect of steroids or drugs, is rapidly evoked by PCN acting on the liver, directly. The concept of a set of genes coordinately controlled to maintain homeostasis in parenchymal tissues during toxic stress must now be extended to include the immune system.     

Coactivator binding promotes the specific interaction between ligand and the pregnane X receptor

The pregnane X receptor (PXR) detects the presence of a wide variety of endogenous and xenobiotic compounds, and is a master regulator of the expression of genes central to drug metabolism and excretion. We present the 2.0A crystal structure of the human PXR ligand-binding domain (LBD) in complex with the cholesterol-lowering compound SR12813 and a 25 amino acid residue fragment of the human steroid receptor coactivator-1 (SRC-1) containing one LXXLL motif. PXR crystallizes as a homodimer in the asymmetric unit in this structure and possesses a novel alpha2 helix adjacent to its ligand-binding cavity. The SRC-1 peptide forms two distinct helices and binds adjacent to the ligand-dependent transactivation AF-2 helix on the surface of PXR. In contrast with previous PXR structures, in which SR12813 bound in multiple orientations, the small SR12813 agonist in this structure binds in a single, unique orientation within the receptor's ligand-binding pocket and contacts the AF-2 helix. Thermal denaturation studies reveal that the SR12813 ligand and SRC-1 coactivator peptide each stabilize the LBD of PXR, and that together they exert an additive effect on the stability of the receptor. These results indicate that the binding of coactivator to the surface of PXR limits the ability of this promiscuous receptor to "breathe" and helps to trap a single, active conformation of SR12813. They further reveal that specificity is required for PXR activation.     

Effects of nonylphenol on hepatic testosterone metabolism and the expression of acute phase proteins in winter flounder (Pleuronectes americanus): comparison to the effects of Saint John's Wort

4-Nonylphenol (4-NP), a major by-product of alkylphenol ethoxylates, is used in several industries and as a consequence is quite common in rivers, estuaries and other aquatic environments that receive sewage discharges or are near offshore oil platforms. 4-NP is an environmental estrogen that also binds human and rodent Pregnane X-receptor (PXR), the orphan nuclear receptor that controls the expression of several detoxication genes in mammals, including several CYP3A and CYP2B family members. These P450s preferentially hydroxylate testosterone in the 6beta- and 16beta-positions, respectively. In this study, the effects of 4-NP on testosterone metabolism and hepatic CYP3A induction were compared to the effects of St. John's Wort (SJW), a well established mammalian PXR agonist, in winter flounder. Male winter flounder (Pleuronectes americanus) were injected with 100 mg/kg/day 4-NP or 500 mg/kg/day SJW or both (S and N) every 24 h. Forty-eight hours after the initial injections, flounder were euthanized. Western blots and testosterone 6beta-hydroxylation indicated that CYP3A was increased 50% by 4-NP, but was not affected by SJW. Testosterone 16beta-hydroxylase activity was also significantly increased in flounder treated with 4-NP (2.8 x), but not with SJW. This is not consistent with our hypothesis that both SJW and 4-NP would induce CYP3A. Subtractive hybridization was performed between control and 4-NP treated hepatic mRNA samples to isolate differentially expressed genes. Subtractive hybridization indicated that several acute phase proteins were altered by 4-NP. Quantitative real-time PCR (Q-PCR) confirmed 4-NP altered the expression of complement components C8b, cathepsin L, C-type lectin domain, FK506 binding protein 2 precursor (FKBP2) and an EST (expressed sequence tag). SJW and 4-NP treated flounder demonstrated similar induction profiles for the EST, cathepsin L and FKBP2, suggesting that SJW was at a sufficient dose to alter gene expression but not induce P450s. In conclusion, testosterone hydroxylase activity and Western blots indicate that SJW did not activate detoxication pathways in a similar manner to 4-NP.