Novel P450(17alpha) inhibitors: 17-(2'-oxazolyl)- and 17-(2'-thiazolyl)-androstene derivatives

Twelve 17-(2'-oxazolyl)- and 17-(2'-thiazolyl)-androsta-5,16-diene derivatives were designed and synthesized from 3 beta-acetoxy-pregna-5,16-dien-20-one (1b) as inhibitors of 17 alpha-hydroxylase-C(17,20)-lyase (P450(17 alpha)). Potent inhibitors of this enzyme could be of value as treatment of prostate cancer. Two substituents (methyl and phenyl) were introduced either at their 4'- or 5'-position in order to investigate their structure-activity relationship. Due to the 16,17-double bond, 17-thiazoles were generally obtained in low yield. The pharmacological results showed that the compounds containing 17-(2'-oxazolyl) (14c) and 17-(2'-thiazolyl) (8c) (41.5%) demonstrated reasonable inhibition against P450(17 alpha). Their 3-acetate (13c and 7c) were less potent than their 3-OH counterparts. The introduction of a phenyl or methyl group generally decreased inhibitory activity. Surprisingly, 17-(5'-methyl-2'-thiazolyl) (12a) was the most potent compound in this series and was almost as potent as L-39, which has good antitumor activity.     

Effects of 17-hydroxyprogesterone caproate (17-OHPC) administration to pregnant squirrel monkeys (Saimiri boliviensis boliviensis)

Poor reproductive performance of the squirrel monkey (Saimiri boliviensis boliviensis) in captivity and a relative progesterone (P) deficiency in pregnancy have been reported. Since premature births may contribute to pregnancy wastage, we attempted to measure the effectiveness of 17-hydroxyprogesterone caproate (17-OHPC) treatment of pregnant squirrel monkeys to prevent early deliveries. Based on clearance studies of nonpregnant animals, 25 mg of 17-OHPC was administered at 6-day intervals to a test group of 31 pregnant monkeys while the control group of 29 received saline. Venous blood was obtained at 6- to 12-day intervals for measurement of 17-hydroxyprogesterone (17-OHP), P, 17-B estradiol (E), and androstenedione (A), and dihydroepiandrosterone (DHEA) levels by radioimmunoassays. The treated group had a significant increase in serum 17-OHP (P < 0.001), P (P < 0.01), and DHEA (P < 0.05) levels compared to controls. The numbers of live births, stillbirths, or neonatal deaths did not differ significantly between groups. Although 17-OHPC administration appeared to increase P and 17-OHP levels, this did not alter the duration of pregnancy nor delay the onset of labor. A significant fall in 17-OHP, P, and E levels was observed 6–12 days before delivery.     

A steroid structure which refutes the isolation of C(17)-C(20) rotational isomers

Isolation of two C(17)-C(20) rotamers of 20-methyl-20-(2-hydroxy-ethoxy)-5-pregnene-3β, 17α-diol has been reported. X-Ray analysis of a diacetate derivative of one of the “rotamers” shows that the actual structure is 3β-acetoxy-17aα-(2-acetoxyethoxy)-17α,17aβ-dimethyl-D-homo-5-androsten-17β-ol (C₂₈H₄₄O₆). Thus, although this investigation refutes the existence of C(17)-C(20) rotamers, it suggests a possible new pathway for D-homo steroid synthesis.     

Effects of interleukin 17A (IL-17A) neutralization on murine hepatitis virus (MHV-A59) infection

Mice infected with mouse hepatitis virus A59 (MHV-A59) develop hepatitis and autoantibodies (autoAb) to liver and kidney fumarylacetoacetate hydrolase (FAH), a fact closely related to the release of alarmins such as uric acid and/or high-mobility group box protein 1 (HMGB1). We studied the effect of neutralizing monoclonal antibodies (MAb) against IL-17A in our model of mouse MHV-A59-infection. MAb anti-IL-17F and anti-IFNγ were used to complement the study. Results showed that transaminase levels markedly decreased in MHV-A59-infected mice treated with MAb anti-IL-17A whereas plasmatic Ig concentration sharply increased. Conversely, MAb anti-IL-17F enhanced transaminase liberation and did not affect Ig levels. Serum IFNγ was detected in mice infected with MHV-A59 and its concentration increased after MAb anti-IL-17A administration. Besides, MAb anti-IFNγ greatly augmented transaminase plasmatic levels. IL-17A neutralization did not affect MHV-A59-induction of HMGB1 liberation and slightly augmented plasmatic uric acid concentration. However, mice treated with the MAb failed to produce autoAb to FAH. The above results suggest a reciprocal regulation of Th1 and Th17 cells acting on the different MHV-A59 effects. In addition, it is proposed that IL-17A is involved in alarmins adjuvant effects leading to autoAb expression.     

Interleukin (IL)-17 enhances prostaglandin F(2 alpha)-stimulated IL-6 synthesis in osteoblasts

We previously showed that prostaglandin F(2alpha) (PGF(2alpha)) and endothelin-1 (ET-1) induce interleukin (IL)-6 through the activation of protein kinase C-dependent p44/p42 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells. It has recently been reported that tumor necrosis factor-alpha-induced IL-6 synthesis is amplified by IL-17 in these cells. In the present study, we investigated the effect of IL-17 on the IL-6 synthesis stimulated by PGF(2alpha) in MC3T3-E1 cells. IL-17 significantly enhanced the PGF(2alpha)-induced IL-6 synthesis in a dose-dependent manner in the range between 0.1 and 10 ng/ml. IL-17 also enhanced the IL-6 synthesis stimulated by 12- O -tetradecanoylphorbol-13-acetate, a direct activator of protein kinase C. In addition, IL-17 amplified the IL-6 synthesis induced by ET-1. However, IL-17 hardly affected the phosphorylation of p44/p42 MAP kinase induced by PGF(2alpha) or ET-1. These results strongly suggest that IL-17 enhances the IL-6 synthesis stimulated by PGF(2alpha) as well as ET-1 in osteoblasts, and that the effect is exerted at a point downstream from p44/p42 MAP kinase.     

Efficient synthesis of 17-acetyl-13-(p-bromophenyl)-3-methoxy-11,11-bis(methoxycarbonyl)gona-1,3,5(10)-trienes

We described an efficient synthesis of (8β,9β,14β)-17β-acetyl-13β-p-bromophenyl-11,11-di(methoxycarbonyl)-3-methoxygona-1,3,5(10)-triene, (8β,9α,14β)-17β-acetyl-13β-p-bromophenyl-11,11-di(methoxycarbonyl)-3-methoxygona-1,3,5(10)-triene, (8β,9β,14β)-13 β-p-bromophenyl-11,11-di(methoxycarbonyl)-17β-(2-hydroxyethyl)-3-methoxygona-1,3,5(10)-triene, and (8β,9β,14β)-13β-p-bromophenyl-11,11-di(methoxycarbonyl)-17β-(2-oxoxyethyl)-3-methoxygona-1,3,5(10)-triene in five or six steps from 1-iodo-4-methoxybenzocyclobutene and readily available materials.     

Interaction of steroids with adrenal cytochrome P-450 (P-450(17)alpha,lyase) in liposome membranes

Purified cytochrome P-450(17)alpha,lyase from guinea-pig adrenal microsomes, which catalyzes progesterone 17 alpha-hydroxylation and sequentially C17-C20 bond cleavage of the 17 alpha-hydroxyprogesterone, was successfully incorporated into liposomal membranes composed of only phosphatidylcholine or of a phospholipid mixture of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine at a molar ratio of 5:3:1. Although the purified P-450(17)alpha,lyase was readily converted into P-420 in the detergent-solubilized system without substrates, the P-450 embedded in the liposomal membranes was found to be quite stable without the substrates. Using the P-450(17)alpha,lyase-proteoliposomes, the interaction of steroids with P-450(17)alpha,lyase was studied for progesterone, 17 alpha-hydroxyprogesterone and androstenedione in the liposomal system by optical difference spectroscopy and by equilibrium dialysis. The partition coefficients of steroids between the aqueous phase and the liposomal membranes were determined by the equilibrium dialysis. They were about 1.4-1.6-times higher in phosphatidylcholine liposomes than in the liposomes of the lipid mixture. The dissociation constants of the P-450-steroid complexes were calculated from the apparent dissociation constants using the partition coefficients for the situation where the substrate-binding site faces the lipid phase of the membranes or where it faces the aqueous phase. The dissociation constant in the former case was not affected by the lipid composition. These results suggest that P-450(17)alpha,lyase might interact only with the substrates in the lipid phase of the liposomal membranes.     

Natural killer (NK) cells are present in mice with severe combined immunodeficiency (scid)

Spleen cells from C.B- 17 scid mice with severe combined immunodeficiency disease exhibit natural killer cell (NK) activity against YAC lymphoma targets in a standard 4-hr 51Cr release assay. The cytolytic activity is demonstrable only at high effector to target ratios but can be augmented at least sevenfold by the interferon inducer poly I:C. The pattern of target lysis is specific, because splenocytes from poly I:C-primed C.B-17 scid mice lyse NK-sensitive YAC cells and not the insensitive P815 mastocytoma. The presence of several NK-associated antigens on C.B-17 scid splenocytes was tested by pretreating cells with the appropriate antiserum plus complement before testing for NK activity. The results indicate that a proportion of NK effectors in C.B-17 scid mice bear surface NK 2.1 and Asialo GM1 but are negative for Thy-1.     

A Cytotoxic Antibody Recognizing Lacto-N-fucopentaose I (LNFP I) on Human Induced Pluripotent Stem (hiPS) Cells

We have generated a mouse monoclonal antibody (R-17F, IgG1 subtype) specific to human induced pluripotent stem (hiPS)/embryonic stem (ES) cells by using a hiPS cell line as an antigen. Triple-color confocal immunostaining images of hiPS cells with R-17F indicated that the R-17F epitope was expressed exclusively and intensively on the cell membranes of hiPS cells and co-localized partially with those of SSEA-4 and SSEA-3. Lines of evidence suggested that the predominant part of the R-17F epitope was a glycolipid. Upon TLC blot of total lipid extracts from hiPS cells with R-17F, one major R-17F-positive band was observed at a slow migration position close to that of anti-blood group H1(O) antigen. MALDI-TOF-MS and MSⁿ analyses of the purified antigen indicated that the presumptive structure of the R-17F antigen was Fuc-Hex-HexNAc-Hex-Hex-Cer. Glycan microarray analysis involving 13 different synthetic oligosaccharides indicated that R-17F bound selectively to LNFP I (Fucα1–2Galβ1–3GlcNAcβ1–3Galβ1–4Glc). A critical role of the terminal Fucα1–2 residue was confirmed by the selective disappearance of R-17F binding to the purified antigen upon α1–2 fucosidase digestion. Most interestingly, R-17F, when added to hiPS/ES cell suspensions, exhibited potent dose-dependent cytotoxicity. The cytotoxic effect was augmented markedly upon the addition of the secondary antibody (goat anti-mouse IgG1 antibody). R-17F may be beneficial for safer regenerative medicine by eliminating residual undifferentiated hiPS cells in hiPS-derived regenerative tissues, which are considered to be a strong risk factor for carcinogenesis.     

17 beta-(cyclopropylamino)-androst-5-en-3 beta-ol, a selective mechanism-based inhibitor of cytochrome P450(17 alpha) (steroid 17 alpha-hydroxylase/C17-20 lyase)

A new compound, 17 beta-(cyclopropylamino)-androst-5-en-3 beta-ol, MDL 27,302, has been designed and synthesized as a mechanism-based inhibitor of cytochrome P450(17 alpha). The time-dependent inactivation of human testicular P450(17 alpha) is irreversible by dialysis and requires the cofactor, NADPH; Kiapp. 90 nM (determined on cynomolgous monkey testis enzyme). Inactivation was not affected by the nucleophile DTT, suggesting retention of the inhibitor in the enzyme active site during the inactivation process. Inhibition is specific to the cyclopropylamino compound, since the isopropylamino- and cyclobutylamino-analogs were not inhibitory. Enzymatic specificity of MDL 27,302 for P450(17 alpha) was demonstrated by its failure to inhibit steroid 21-hydroxylase and the cholesterol side chain cleavage enzyme (P450scc). Both the 17 alpha-hydroxylase and C17-20 lyase activities of cytochrome P450(17 alpha) of human testis microsomes were inhibited by MDL 27,302.     

Increased interleukin (IL)-8 and decreased IL-17 production in chronic obstructive pulmonary disease (COPD) provoked by cigarette smoke

Recently, involvement of IL-17 in development of COPD has been noticed. Unlike IL-8, the role of IL-17 in COPD remains controversial. In order to further understand mechanisms in cigarette smoke (CS) induced COPD, we investigated IL-17 and IL-8 levels in different stages of COPD patients, and time courses of IL-17 and IL-8 release in CS induced COPD rats. A total of 73 elderly patients with COPD and 31 healthy volunteers were recruited in the study. IL-17 and IL-8 levels in the sputum and plasma were measured, and number of differential cells was counted. A newly developed CS induced rat COPD model was employed to study time courses of IL-17 and IL-8 release and nucleated cell accumulation. The results showed that IL-8 levels in the sputum of severe COPD patients were elevated by 16.5-fold, but IL-17 levels were reduced by 4.8-fold. While IL-8 correlated with neutrophils, IL-17 correlated with monocytes and lymphocytes. Similarly, level of IL-8 was increased, but IL-17 was decreased in the bronchoalveolar lavage fluid (BALF) of CS rats. Time course study showed that increased IL-8 production in the BALF initiated at 6 weeks, but decreased IL-17 production started at 10 weeks following CS exposure. In conclusion, increased IL-8 level in COPD patients appears mainly secreted from neutrophils and macrophages, whereas decreased IL-17 level seems resulted from reduced number of monocytes or damaged epithelial cells. Increased IL-8 (a proinflammatory cytokine) secretion and decreased IL-17 (a protective cytokine of airways) release can both contribute to development of COPD.     

Induction of 17 alpha-hydroxylase (cytochrome P-450(17)alpha) activity by adrenocorticotropin in bovine adrenocortical cells maintained in monolayer culture

Using bovine adrenocortical cells in monolayer culture it has been shown that treatment with adrenocorticotropin (ACTH) causes a dramatic increase in 17 alpha-hydroxylase activity. In postmitochondrial supernatant fractions (PMS) prepared from cells maintained in culture, there was a 15-fold increase in 17 alpha-hydroxylase activity 36 h following initiation of ACTH treatment compared with the activity measured in PMS prepared from control cells. In the continued presence of ACTH, 17 alpha-hydroxylase activity declined; however, even after 60 h of exposure to ACTH, 17 alpha-hydroxylase activity was eight times higher than that present in control cells. The dramatic increase in 17 alpha-hydroxylase activity provides an explanation for the previously observed phenomenon that following initiation of ACTH treatment of bovine adrenocortical cells in monolayer culture there is a shift in the pattern of corticosteroid secretion from approximately equal amounts of cortisol and corticosterone to almost exclusively cortisol. Thus, the modulation of 17 alpha-hydroxylase activity by ACTH action appears to serve a key regulatory role in the pattern of corticosteroid production. Soluble cytosolic factors apparently do not participate in the regulation of 17 alpha-hydroxylase activity in the bovine adrenal cortex. Increases in the magnitude of substrate-induced absorbance changes are indicative that the increase in 17 alpha-hydroxylase activity is due, at least in part, to an elevation of cytochrome P-450(17)alpha synthesis.     

Differential regulation of central nervous system autoimmunity by T(H)1 and T(H)17 cells

Multiple sclerosis is an inflammatory, demyelinating disease of the central nervous system (CNS) characterized by a wide range of clinical signs. The location of lesions in the CNS is variable and is a crucial determinant of clinical outcome. Multiple sclerosis is believed to be mediated by myelin-specific T cells, but the mechanisms that determine where T cells initiate inflammation are unknown. Differences in lesion distribution have been linked to the HLA complex, suggesting that T cell specificity influences sites of inflammation. We demonstrate that T cells that are specific for different myelin epitopes generate populations characterized by different T helper type 17 (T(H)17) to T helper type 1 (T(H)1) ratios depending on the functional avidity of interactions between TCR and peptide-MHC complexes. Notably, the T(H)17:T(H)1 ratio of infiltrating T cells determines where inflammation occurs in the CNS. Myelin-specific T cells infiltrate the meninges throughout the CNS, regardless of the T(H)17:T(H)1 ratio. However, T cell infiltration and inflammation in the brain parenchyma occurs only when T(H)17 cells outnumber T(H)1 cells and trigger a disproportionate increase in interleukin-17 expression in the brain. In contrast, T cells showing a wide range of T(H)17:T(H)1 ratios induce spinal cord parenchymal inflammation. These findings reveal critical differences in the regulation of inflammation in the brain and spinal cord.     

Tumor necrosis factor-alpha convertase (ADAM17) mediates regulated ectodomain shedding of the severe-acute respiratory syndrome-coronavirus (SARS-CoV) receptor, angiotensin-converting enzyme-2 (ACE2)

Angiotensin-converting enzyme-2 (ACE2) is a critical regulator of heart function and a cellular receptor for the causative agent of severe-acute respiratory syndrome (SARS), SARS-CoV (coronavirus). ACE2 is a type I transmembrane protein, with an extracellular N-terminal domain containing the active site and a short intracellular C-terminal tail. A soluble form of ACE2, lacking its cytosolic and transmembrane domains, has been shown to block binding of the SARS-CoV spike protein to its receptor. In this study, we examined the ability of ACE2 to undergo proteolytic shedding and investigated the mechanisms responsible for this shedding event. We demonstrated that ACE2, heterologously expressed in HEK293 cells and endogenously expressed in Huh7 cells, undergoes metalloproteinase-mediated, phorbol ester-inducible ectodomain shedding. By using inhibitors with differing potency toward different members of the ADAM (a disintegrin and metalloproteinase) family of proteases, we identified ADAM17 as a candidate mediator of stimulated ACE2 shedding. Furthermore, ablation of ADAM17 expression using specific small interfering RNA duplexes reduced regulated ACE2 shedding, whereas overexpression of ADAM17 significantly increased shedding. Taken together, these data provided direct evidence for the involvement of ADAM17 in the regulated ectodomain shedding of ACE2. The identification of ADAM17 as the protease responsible for ACE2 shedding may provide new insight into the physiological roles of ACE2.     

Structural Determinants for Binding of Sorting Nexin 17 (SNX17) to the Cytoplasmic Adaptor Protein Krev Interaction Trapped 1 (KRIT1)

Sorting nexin 17 (SNX17) is a member of the family of cytoplasmic sorting nexin adaptor proteins that regulate endosomal trafficking of cell surface proteins. SNX17 localizes to early endosomes where it directly binds NPX(Y/F) motifs in the cytoplasmic tails of its target receptors to mediate their rates of endocytic internalization, recycling, and/or degradation. SNX17 has also been implicated in mediating cell signaling and can interact with cytoplasmic proteins. KRIT1 (Krev interaction trapped 1), a cytoplasmic adaptor protein associated with cerebral cavernous malformations, has previously been shown to interact with SNX17. Here, we demonstrate that SNX17 indeed binds directly to KRIT1 and map the binding to the second Asn-Pro-Xaa-Tyr/Phe (NPX(Y/F)) motif in KRIT1. We further characterize the interaction as being mediated by the FERM domain of SNX17. We present the co-crystal structure of SNX17-FERM with the KRIT1-NPXF2 peptide to 3.0 Å resolution and demonstrate that the interaction is highly similar in structure and binding affinity to that between SNX17 and P-selectin. We verify the molecular details of the interaction by site-directed mutagenesis and pulldown assay and thereby confirm that the major binding site for SNX17 is confined to the NPXF2 motif in KRIT1. Taken together, our results verify a direct interaction between SNX17 and KRIT1 and classify KRIT1 as a SNX17 binding partner.     

Inhibition of 17,20(17-hydroxyprogesterone)-lyase by progesterone

¹⁴C-17-Hydroxyprogesterone was incubated with 7000 × g × 20 min supernatants of rat testis homogenates in the presence of various concentrations of ³H-progesterone, both under conditions where metabolism would take place and where it would be prevented. When metabolism was prevented, the ratio of progesterone to 17-hydroxyprogesterone in the microsomal fraction was 3 times that which was added to the incubation medium.Progesterone competitively inhibited 17,20-lyase action on added 17-hydroxyprogesterone but not on 17-hydroxyprogesterone formed from the added progesterone. The rate of formation of 17-hydroxyprogesterone from progesterone, however, was inhibited by added 17-hydroxyprogesterone. The results indicate that there is no free exchange of an intermediate between progesterone and androstenedione with the soluble fraction, either inside or outside the microsomal vesicle. The limited exchange with 17-hydroxyprogesterone in solution probably represents exchange with an enzyme-bound intermediate.     

Mitochondria play an important role in 17beta-estradiol attenuation of H(2)O(2)-induced rat endothelial cell apoptosis

Studies have shown salutary effects of 17beta-estradiol following trauma-hemorrhage on different cell types. 17beta-Estradiol also induces improved circulation via relaxation of the aorta and has an anti-apoptotic effect on endothelial cells. Because mitochondria play a pivotal role in apoptosis, we hypothesized that 17beta-estradiol will maintain mitochondrial function and will have protective effects against H(2)O(2)-induced apoptosis in endothelial cells. Endothelial cells were isolated from rats' aorta and cultured in the presence or absence of H(2)O(2), a potent inducer of apoptosis. In additional studies, endothelial cells were pretreated with 17beta-estradiol. Flow cytometry analysis revealed H(2)O(2)-induced apoptosis in 80.9% of endothelial cells; however, prior treatment of endothelial cells with 17beta-estradiol resulted in an approximately 40% reduction in apoptosis. This protective effect of 17beta-estradiol was abrogated when endothelial cells were cultured in the presence ICI-182780, indicating the involvement of estrogen receptor (ER). Fluorescence microscopy revealed a 17beta-estradiol-mediated attenuation of H(2)O(2)-induced mitochondrial condensation. Western blot analysis demonstrated that H(2)O(2)-induced cytochrome c release from mitochondrion to cytosol and the activation of caspase-9 and -3 were decreased by 17beta-estradiol. These findings suggest that 17beta-estradiol attenuated H(2)O(2)-induced apoptosis via ER-dependent activation of caspase-9 and -3 in rat endothelial cells through mitochondria.     

A brief history of T(H)17, the first major revision in the T(H)1/T(H)2 hypothesis of T cell-mediated tissue damage

For over 35 years, immunologists have divided T-helper (T(H)) cells into functional subsets. T-helper type 1 (T(H)1) cells-long thought to mediate tissue damage-might be involved in the initiation of damage, but they do not sustain or play a decisive role in many commonly studied models of autoimmunity, allergy and microbial immunity. A major role for the cytokine interleukin-17 (IL-17) has now been described in various models of immune-mediated tissue injury, including organ-specific autoimmunity in the brain, heart, synovium and intestines, allergic disorders of the lung and skin, and microbial infections of the intestines and the nervous system. A pathway named T(H)17 is now credited for causing and sustaining tissue damage in these diverse situations. The T(H)1 pathway antagonizes the T(H)17 pathway in an intricate fashion. The evolution of our understanding of the T(H)17 pathway illuminates a shift in immunologists' perspectives regarding the basis of tissue damage, where for over 20 years the role of T(H)1 cells was considered paramount.