Kamalesh K. Sharma

Identification of Ligands for DAF-12 that Govern Dauer Formation and Reproduction in C. elegans

In response to environmental and dietary cues, the C. elegans orphan nuclear receptor, DAF-12, regulates dauer diapause, reproductive development, fat metabolism, and life span. Despite strong evidence for hormonal control, the identification of the DAF-12 ligand has remained elusive. In this work, we identified two distinct 3-keto-cholestenoic acid metabolites of DAF-9, a cytochrome P450 involved in hormone production, that function as ligands for DAF-12. At nanomolar concentrations, these steroidal ligands (called dafachronic acids) bind and transactivate DAF-12 and rescue the hormone deficiency of daf-9 mutants. Interestingly, DAF-9 has a biochemical activity similar to mammalian CYP27A1 catalyzing addition of a terminal acid to the side chain of sterol metabolites. Together, these results define the first steroid hormones in nematodes as ligands for an invertebrate orphan nuclear receptor and demonstrate that steroidal regulation of reproduction, from biology to molecular mechanism, is conserved from worms to humans.

Identification of the nuclear receptor DAF-12 as a therapeutic target in parasitic nematodes

Nematode parasitism is a worldwide health problem resulting in malnutrition and morbidity in over 1 billion people. The molecular mechanisms governing infection are poorly understood. Here, we report that an evolutionarily conserved nuclear hormone receptor signaling pathway governs development of the stage 3 infective larvae (iL3) in several nematode parasites, including Strongyloides stercoralis, Ancylostoma spp., and Necator americanus. As in the free-living Caenorhabditis elegans, steroid hormone-like dafachronic acids induced recovery of the dauer-like iL3 in parasitic nematodes by activating orthologs of the nuclear receptor DAF-12. Moreover, administration of dafachronic acid markedly reduced the pathogenic iL3 population in S. stercoralis, indicating the potential use of DAF-12 ligands to treat disseminated strongyloidiasis. To understand the pharmacology of targeting DAF-12, we solved the 3-dimensional structure of the S. stercoralis DAF-12 ligand-binding domain cocrystallized with dafachronic acids. These results reveal the molecular basis for DAF-12 ligand binding and identify nuclear receptors as unique therapeutic targets in parasitic nematodes.

Abiraterone Inhibits 3β-Hydroxysteroid Dehydrogenase: A Rationale for Increasing Drug Exposure in Castration-Resistant Prostate Cancer

Purpose: Treatment with abiraterone (abi) acetate prolongs survival in castration-resistant prostate cancer (CRPC). Resistance to abi invariably occurs, probably due in part to upregulation of steroidogenic enzymes and/or other mechanisms that sustain dihydrotestosterone (DHT) synthesis, which raises the possibility of reversing resistance by concomitant inhibition of other required steroidogenic enzymes. On the basis of the 3β-hydroxyl, Δ5-structure, we hypothesized that abi also inhibits 3β-hydroxysteroid dehydrogenase/isomerase (3βHSD), which is absolutely required for DHT synthesis in CRPC, regardless of origins or routes of synthesis. Experimental Design: We tested the effects of abi on 3βHSD activity, androgen receptor localization, expression of androgen receptor–responsive genes, and CRPC growth in vivo. Results: Abi inhibits recombinant 3βHSD activity in vitro and endogenous 3βHSD activity in LNCaP and LAPC4 cells, including conversion of [3H]-dehydroepiandrosterone (DHEA) to Δ4-androstenedione, androgen receptor nuclear translocation, expression of androgen receptor–responsive genes, and xenograft growth in orchiectomized mice supplemented with DHEA. Abi also blocks conversion of Δ5-androstenediol to testosterone by 3βHSD. Abi inhibits 3βHSD1 and 3βHSD2 enzymatic activity in vitro; blocks conversion from DHEA to androstenedione and DHT with an IC50 value of less than 1 μmol/L in CRPC cell lines; inhibits androgen receptor nuclear translocation; expression of TMPRSS2, prostate-specific antigen, and FKBP5; and decreases CRPC xenograft growth in DHEA-supplemented mice. Conclusions: We conclude that abi inhibits 3βHSD-mediated conversion of DHEA to active androgens in CRPC. This second mode of action might be exploited to reverse resistance to CYP17A1 inhibition at the standard abi dose by dose-escalation or simply by administration with food to increase drug exposure. Clin Cancer Res; 18(13); 3571–9. ©2012 AACR.

Deoxycorticosterone inactivation by AKR1C3 in human mineralocorticoid target tissues

Aldosterone is the principal endogenous mineralocorticoid in humans and regulates salt and water homeostasis. Cortisol, the major glucocorticoid, has high affinity for the mineralocorticoid receptor; however, 11beta-hydroxysteroid dehydrogenase type 2 converts cortisol to the inactive steroid cortisone in aldosterone target cells of the kidney, thus limiting the mineralocorticoid action of cortisol. Deoxycorticosterone (DOC) binds to the mineralocorticocoid receptor with high affinity and circulates at concentrations comparable to aldosterone. Severe DOC excess as is seen in 17alpha- and 11beta-hydroxylase deficiencies causes hypertension, and moderate DOC overproduction in late pregnancy is associated with hypertension. Here, we demonstrate that DOC is inactivated by the 20-ketosteroid reductase activity of the human AKR1C3 isozyme. Immunohistochemical analyses demonstrate that AKR1C3 is expressed in the mineralocorticoid-responsive epithelial cells of the renal cortical and medullary collecting ducts, as well as the colon. Our findings suggest that AKR1C3 protects the mineralocorticoid receptor from activation by DOC in mineralocorticoid target cells of the kidney and colon, analogous to cortisol inactivation by 11beta-hydroxysteroid dehydrogenase type 2.

Synthesis and activity of dafachronic acid ligands for the C. elegans DAF-12 nuclear hormone receptor

The nuclear hormone receptor DAF-12 from Caenorhabditis elegans is activated by dafachronic acids, which derive from sterols upon oxidation by DAF-9, a cytochrome P450. DAF-12 activation is a critical checkpoint in C. elegans for acquisition of reproductive competence and for entry into adulthood rather than dauer diapause. Previous studies implicated the (25S)-Delta(7)-dafachronic acid isomer as the most potent compound, but the (25S)-Delta(4)-isomer was also identified as an activator of DAF-12. To explore the tolerance of DAF-12 for structural variations in the ligand and to enable further studies requiring large amounts of ligands for DAF-12 and homologs in other nematodes, we synthesized (25R)- and (25S)-isomers of five dafachronic acids differing in A/B-ring configurations. Both the (25S)- and (25R)-Delta(7)-dafachronic acids are potent transcriptional activators in a Gal4-transactivation assay using HEK-293 cells, with EC(50) values of 23 and 33 nm, respectively, as are (25S)- and (25R)-Delta(4)-dafachronic acids, with EC(50) values of 23 and 66 nm, respectively. The (25S)- and (25R)-Delta(5)-isomers were much less potent, with EC(50) values approaching 1000 nm, and saturated 5alpha- and 5beta-dafachronic acids showed mostly intermediate potencies. Rescue assays using daf- 9-null mutants confirmed the results from transactivation experiments, but this in vivo assay accentuated the greater potencies of the (25S)-epimers, particularly for the (25S)-Delta(7)-isomer. We conclude that DAF-12 accommodates a large range of structural variation in ligand geometry, but (25S)-Delta(7)-dafachronic acid is the most potent and probably biologically relevant isomer. Potency derives more from the A/B-ring configuration than from the stereochemistry at C-25.

Stable 5,6-Epoxyeicosatrienoic Acid Analog Relaxes Coronary Arteries Through Potassium Channel Activation

5,6-Epoxyeicosatrienoic acid (5,6-EET) is a cytochrome P450 epoxygenase metabolite of arachidonic acid that causes vasorelaxation. However, investigations of its role in biological systems have been limited by its chemical instability. We developed a stable agonist of 5,6-EET, 5-(pentadeca-3(Z),6(Z),9(Z)-trienyloxy)pentanoic acid (PTPA), in which the 5,6-epoxide was replaced with a 5-ether. PTPA obviates chemical and enzymatic hydrolysis. In bovine coronary artery rings precontracted with U46619, PTPA (1 nmol/L to 10 &mgr;mol/L) induced concentration-dependent relaxations, with maximal relaxation of 86±5% and EC50 of 1 &mgr;mol/L. The relaxations were inhibited by the cyclooxygenase inhibitor indomethacin (10 &mgr;mol/L; max relaxation 43±9%); the ATP-sensitive K+ channel inhibitor glybenclamide (10 &mgr;mol/L; max relaxation 49±6%); and the large conductance calcium-activated K+ channel inhibitor iberiotoxin (100 nmol/L; max relaxation 38±6%) and abolished by the combination of iberiotoxin with indomethacin or glybenclamide or increasing extracellular K+ to 20 mmol/L. Whole-cell outward K+ current was increased nearly 6-fold by PTPA (10 &mgr;mol/L), which was also blocked by iberiotoxin. Additionally, we synthesized 5-(pentadeca-6(Z),9(Z)-dienyloxy)pentanoic acid and 5-(pentadeca-3(Z),9(Z)-dienyloxy)pentanoic acid (PDPA), PTPA analogs that lack the 8,9 or 11,12 double bonds of arachidonic acid and therefore are not substrates for cyclooxygenase. The PDPAs caused concentration-dependent relaxations (max relaxations 46±13% and 52±7%, respectively; EC50 1&mgr;mol/L), which were not altered by glybenclamide but blocked by iberiotoxin. These studies suggested that PTPA induces relaxation through 2 mechanisms: (1) cyclooxygenase-dependent metabolism to 5-ether–containing prostaglandins that activate ATP-sensitive K+ channels and (2) activation of smooth muscle large conductance calcium-activated K+ channels. PDPAs only activate large conductance calcium-activated K+ channels.

Why human cytochrome P450c21 is a progesterone 21-hydroxylase

Human cytochrome P450c21 (steroid 21-hydroxylase, CYP21A2) catalyzes the 21-hydroxylation of progesterone (P4) and its preferred substrate 17α-hydroxyprogestrone (17OHP4). CYP21A2 activities, which are required for cortisol and aldosterone biosynthesis, involve the formation of energetically disfavored primary carbon radicals. Therefore, we hypothesized that the binding of P4 and 17OHP4 to CYP21A2 restricts access of the reactive heme-oxygen complex to the C-21 hydrogen atoms, suppressing oxygenation at kinetically more favorable sites such as C-17 and C-16, which are both hydroxylated by cytochrome P450c17 (CYP17A1). We reasoned that expansion of the CYP21A2 substrate-binding pocket would increase substrate mobility and might yield additional hydroxylation activities. We built a computer model of CYP21A2 based principally on the crystal structure of CYP2C5, which also 21-hydroxylates P4. Molecular dynamics simulations indicate that binding of the steroid nucleus perpendicular to the plane of the CYP21A2 heme ring limits access of the heme oxygen to the C-21 hydrogen atoms. Residues L107, L109, V470, I471, and V359 were found to contribute to the CYP21A2 substate-binding pocket. Mutation of V470 and I471 to alanine or glycine preserved P4 21-hydroxylase activity, and mutations of L107 or L109 were inactive. Mutations V359A and V359G, in contrast, acquired 16α-hydroxylase activity, accounting for 40% and 90% of the P4 metabolites, respectively. We conclude that P4 binds to CYP21A2 in a fundamentally different orientation than to CYP17A1 and that expansion of the CYP21A2 substrate-binding pocket allows additional substrate trajectories and metabolic switching.

Inhibition of 3β-hydroxysteroid dehydrogenase by abiraterone as a rationale for dose escalation in castration-resistant prostate cancer.

209 Background: Treatment with abiraterone acetate (abi) increases the survival of men with castration-resistant prostate cancer (CRPC). Resistance to abi invariably occurs, probably due in part to up-regulation of steroidogenic enzymes and/or other mechanisms that sustain the synthesis of dihydrotestosterone (DHT), which raises the possibility of reversing resistance by concomitant inhibition of other required steroidogenic enzymes. The 1000 mg daily abi dose was selected for the phase III trials despite the absence of dose-limiting toxicities at higher doses. Based on the 3β-hydroxyl, Δ5-structure, we hypothesized that abi also inhibits 3β-hydroxysteroid dehydrogenase/isomerase (3βHSD), which is absolutely required for the intratumoral synthesis of DHT in CRPC, regardless of origins or routes of synthesis. METHODS We tested if abi inhibits recombinant 3βHSD2 activity in vitro or endogenous 3βHSD activity in LNCaP and LAPC4 cells, including conversion of [3H]-dehydroepiandrosterone (DHEA) to androstenedione (AD), androgen receptor (AR) nuclear translocation, expression of AR-responsive genes, and LAPC4 xenograft growth in orchiectomized mice supplemented with DHEA. RESULTS Abi has a mixed inhibition pattern of 3βHSD2 in vitro, blocks the conversion from DHEA to AD and DHT with an IC50 of < 1 µM in CRPC cell lines, inhibits AR nuclear translocation and expression of TMPRSS2, and decreases CRPC xenograft growth in DHEA-supplemented mice. CONCLUSIONS Abi blocks 3βHSD enzymatic activity, synthesis of AD and DHT, inhibits the AR-response, and suppresses growth of CRPC cells at concentrations that are clinically achievable. Variable abi inhibition of 3βHSD might account in part for the heterogeneous clinical response to abi. More importantly, 3βHSD inhibition with abi might be clinically harnessed to reverse resistance to CYP17A1 inhibition at the standard dose by dose-escalation, or simply by administration with food to increase drug exposure.