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. Author manuscript; available in PMC: 2024 Oct 26.
Published in final edited form as: Methods Enzymol. 2023 Apr 25;689:39–63. doi: 10.1016/bs.mie.2023.04.001

Steroid 17α-hydroxylase/17, 20-lyase (cytochrome P450 17A1)

F Peter Guengerich 1,*, Kevin D McCarty 1, Yasuhiro Tateishi 1, Lu Liu 1
PMCID: PMC11512470  NIHMSID: NIHMS2030083  PMID: 37802581

Abstract

Cytochrome P450 (P450) 17A1 plays a key role in steroidogenesis, in that this enzyme catalyzes the 17α-hydroxylation of both pregnenolone and progesterone, followed by a lyase reaction to cleave the C-20 land C-21 carbons from each steroid. The reactions are important in the production of both glucocorticoids and androgens. The enzyme is critical in humans but is also a drug target in treatment of prostate cancer. Detailed methods are described for the heterologous expression of human P450 17A1 in bacteria, purification of the recombinant enzyme, reconstitution of the enzyme system in the presence of cytochrome b5, and chromatographic procedures for sensitive analyses of reaction products. Historic assay approaches are reviewed. Some information is also provided about outstanding questions in the research field, including catalytic mechanisms and searches for selective inhibitors.

1. Introduction

Cytochrome P450 (P450, CYP) 17A1 plays a key role in steroidogenesis. Its primary substrates are pregnenolone, the first product of cholesterol utilized in the biosynthesis of steroids, and progesterone, a 2-electron oxidation product of pregnenolone generated by 3β-hydro-xysteroid dehydrogenase/ketosteroid isomerase (3bHSD2) (Fig. 1). The 17α-hydroxy (OH) products are substrates for further oxidation by P450 17A1, a “lyase” reaction that removes two carbons at the 17-position. The 17α-OH steroids are used to produce glucocorticoids (e.g., cortisol), and the lyase products are androgens (or precursors), which are further transformed to estrogens by P450 19A1. The enzyme is expressed in endocrine tissues, and for information on the localization of P450 17A1 in humans and animals, see the Protein Atlas website (https://www.proteinatlas.org/ENSG00000148795-CYP17A1/tissue) (Auchus & Miller, 2015; Guengerich, 2015). Thus, P450 17A1 plays a central role in mammalian steroidogenesis, and >100 single nucleotide variants have been identified in terms of clinical deficiencies (Auchus, 2017; Auchus & Miller, 2015; DeVore & Scott, 2012). Some of these, especially the few that preferentially affect the lyase reaction, lead to sexual identity disorders (Auchus, 2017; Idkowiak et al., 2012; Kok et al., 2010).

Fig. 1.

Fig. 1

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Major reactions catalyzed by P450 17A1. DHEA: dehydroepiandrosterone.

Some minor reactions products are also known (Table 1). Crystal structures of human P450 17A1 are available with each of the four natural substrates (Fig. 1) and with the inhibitory drug abiraterone and its metabolites (DeVore & Scott, 2012; Petrunak et al., 2014; Petrunak et al., 2023).

Table 1.

P450 17A1 reaction products (Yoshimoto et al., 2016).a

Substrate Products
Progesterone 17α-OH progesterone (major)
16α-OH progesterone
21-OH progesterone
Androstenedione (major)
16α,17α-(OH)2 progesterone
6p,16α,17α-(OH)3 progesterone
16-OH androstenedione
Pregnenolone 17α-OH pregnenolone (major)
Pregnenolone 16,17-diene (5,16-androstadien-3β-ol)
16α,17α-(OH)2 pregnenolone (algesterone)
DHEA (major)
16-OH DHEA
X,16,17α-(OH)3 pregnenolone (X unidentified, probably in B ring)
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a

For more on uncommon substrates and minor reactions, see ref. Auchus and Miller (2015), Gonzalez and Guengerich (2017), Yoshimoto et al. (2016).

2. Heterologous expression and purification of human P450 17A1

Large quantities of highly purified human P450 17A1 are required for many in vitro assays and biophysical studies. This protocol below gives a detailed step-by-step procedure to employ a recombinant protein expression system in Escherichia coli to produce large quantities of P450 17A1 and a Ni2+–NTA column chromatography to purify the enzyme.

2.1. Materials and reagents

  1. Codon-optimized pCW (Ori+) plasmid containing P450 17A1 cDNA, with a (His)6 tag on the C-terminus (Kim et al., 2021)

  2. E. coli JM109 cells

  3. Luria-Bertani (LB) agar ampicillin plate

  4. Luria-Bertani (LB) media containing ampicillin (100 μg/mL)

  5. Terrific Broth (TB) media containing ampicillin (100 μg/mL)

  6. Trace element solution (add 0.25 mL per L of culture–composition: 27 g FeCl3·4H2O, 2 g ZnCl2·H2O, 2 g CoCl2·6H2O, 2 g Na2MoO4·2H2O, 1 g CaCl2·H2O, 1 g CuCl2, 0.5 g H3BO3, and 100 mL conc HCl per L) (Sandhu et al., 1994)

  7. 1 M Isopropyl β-d-1-thiogalactopyranoside (IPTG) stock solution

  8. 1 M δ-Aminolevulinic acid stock solution

  9. 2X TES solution (100 mM Tris–HCl (pH 7.4), 0.5 mM EDTA, and 500 mM sucrose)

  10. Chicken egg white lysozyme

  11. Sonification buffer (300 mM potassium phosphate, pH 7.4, 20% glycerol (v/v), 6 mM magnesium acetate, 0.1 mM DTT and 0.1 mM phenylmethylsulfonyl fluoride)

  12. Protease inhibitor cocktail (Roche)

  13. Nickel nitriloacetic acid (Ni2+–NTA) column (1.5 cm × 8 cm)

  14. Buffer A (300 mM potassium phosphate (pH 7.4), 300 mM NaCl, 20% glycerol (v/v), 0.1 mM DTT, and 20 mM imidazole)

  15. Buffer B (300 mM potassium phosphate (pH 7.4), 300 mM NaCl, 20% glycerol (v/v), 0.1 mM DTT, and 50 mM imidazole)

  16. Buffer C (300 mM potassium phosphate (pH 7.4), 300 mM NaCl, 20% glycerol (v/v), 0.1 mM DTT, and 80 mM imidazole)

  17. Buffer D (300 mM potassium phosphate (pH 7.4), 300 mM NaCl, 20% glycerol (v/v), 0.1 mM DTT, and 100 mM imidazole)

  18. Buffer E (300 mM potassium phosphate (pH 7.4), 300 mM NaCl, 20% glycerol (v/v), 0.1 mM DTT, and 150 mM imidazole)

  19. Dialysis buffer (200 mM potassium phosphate (pH 7.4), 20% glycerol (v/v), and 0.1 mM DTT)

  20. Centrifugal Filters (Millipore Amicon Ultra-15, 30 kDa)

  21. Dialysis membranes (Spectrum Labs Spectra/Por 1 6–8 kDa MWCO Standard RC Dry Dialysis Kit)

2.2. Equipment

  1. Incubator (Precision Gravity Convention)

  2. Shaking incubator (ATR Infors HT AJ125TC Multitron Triple-Stacked Floor Incubator)

  3. Spectrophotometer (Thermo Scientific Genesys 10UV)

  4. Centrifuge (Sorvall RC3B Plus)

  5. Freezer (SO-LOW −80 °C U85–22)

  6. Sonicator (Fisherbrand Model 505 Sonic Dismembrator)

  7. Centrifuge (Du Pont Sorvall RC-5B Refrigerated Superspeed)

  8. Ultracentrifuge (Beckman Coulter Optima L-90K)

2.3. Protocols

Step-by-step procedure:

  1. Transform pCW (Ori+) P450 17A1 plasmid into E. coli JM109 cells. Incubate the cells on a LB agar ampicillin plate in an incubator (Precision Gravity Convention Incubator) at 37 °C.

  2. Inoculate Luria-Bertani (LB) media containing ampicillin (100 μg/mL) with one colony from step 1 under sterile conditions.

  3. Shake and incubate inoculated LB media at 250 rpm and 37 °C overnight in a shaking incubator (ATR Infors HT AJ125TC Multitron Triple-Stacked Floor Incubator Shaker).

  4. Inoculate Terrific Broth (TB) media containing ampicillin (100 μg/mL) with 1% (v/v) of pre-culture from step 3% and 0.025% (v/v) of trace elements.

  5. Shake and incubate TB media at 250 rpm and 37 °C in a shaking incubator.

  6. Monitor optical density at 600 nm (OD600) of the TB media in a spectrophotometer.

  7. Induce the cells by addition of isopropyl β-d-1-thiogalactopyranoside (IPTG) (1.0 mM) and Δ-aminolevulinic acid (1.0 mM), after OD600 reaches ~0.3 (see note 1).

  8. Shake and incubate TB media at 200 rpm and 30 °C for 24–30 h (see note 2).

  9. Harvest cells by spinning TB media at 4 °C, 5000 g for 30 min in a centrifuge (Sorvall RC 3B Plus).

  10. Resuspend pellet in 2 × TES solution.

  11. Add the same volume of cold water as 2 × TES solution.

  12. Treat the cell with chicken egg white lysozyme (75 μg/mL) for 30 min at 4 °C.

  13. Spin the mixture at 5000g for 30 min in a centrifuge (Sorvall RC3B Plus).

  14. Store the pellet in freezer (SO-LOW −80 °C U85–22) for later use.

  15. Thaw cell pellet on ice.

  16. Resuspend cell pellet in sonification buffer with protease inhibitor cocktail (Roche, 1 pill per 50 mL).

  17. Sonicate mixture on ice at 45% amplitude for 10–15 min with 15 s pulse on and 45 s pulse off with a sonicator (Fisherbrand Model 505 Sonic Dismembrator).

  18. Spin the homogenized solution at 10,000g and 4 °C for 50 min in a centrifuge (Du Pont Sorvall RC-5B Refrigerated Superspeed).

  19. Spin the supernatant at 100,000g and 4 °C for 1 h in an ultracentrifuge (Beckman Coulter Optima L-90K).

  20. Equilibrate the nickel nitriloacetic acid (Ni2+–NTA) column (1.5 cm × 8 cm) with buffer A during steps 18 and 19.

  21. Load the supernatant from step 19 onto the Ni2+–NTA column (see note 3).

  22. Wash the Ni2+–NTA column with 10 column volumes of buffer A.

  23. Wash the Ni2+–NTA column with ≥ 2 column volumes of buffer B.

  24. Wash the Ni2+–NTA column with ≥ 2 column volumes of buffer C.

  25. Wash the Ni2+–NTA column with ≥ 2 column volumes of buffer D.

  26. Elude the Ni2+–NTA column with ≥ 2 column volumes of buffer E (see note 4).

  27. Scan fractions at 417 nm with a spectrophotometer (Thermo Scientific Genesys 10UV).

  28. Pool fractions with absorbance at 417 nm above baseline (step 27)–these fractions can also be analyzed for purity by SDS gel electrophoresis (7.5% acrylamide gel) before pooling.

  29. Concentrate sample using centrifugal filters (Millipore Amicon Ultra-15 Centrifugal Filter 30 kDa) by spinning at 3000g and 4 °C.

  30. Dialyze sample (Spectrum Labs Spectra/Por 1 6–8 kDa MWCO Standard RC Dry Dialysis Kit) against dialysis buffer overnight at 4 °C.

  31. Assay P450 content using carbon monoxide and sodium dithionite (Guengerich et al., 2009; Guengerich, 2014)

  32. Store enzyme in ultracold freezer (SO LOW −80 °C U85–22).

2.4. Notes

  1. Optical density of the E. coli population at 600 nm is reached in ~4 h.

  2. Optimized expression was achieved at 200 rpm and 30 °C after 29 h.

  3. Steps 21–26 should be carried out at 4 °C.

  4. P450 h17A1 is a very prominent red band on a bluish-green Ni2+–NTA column. The band might start to travel downward as early as during buffer B wash. Collect fraction sometimes during buffer C or buffer D wash or whenever the red band is close to being eluted.

2.5. Safety considerations and standards

While handling bacteria, wear a lab coat and gloves, sanitize the bench area with 70% ethanol (v/v) before and afterwards, and discard any contaminants in a biohazards waste bin.

3. Assays

Steroids often exert potent biological activities at low physiological concentrations, requiring sensitive analytical methods to detect them.

3.1. Model substrates

No substrates have been found that can be used for continuous spectral assays. The dye Nile Red is a substrate (Guengerich et al., 2019; Lampe et al., 2008) but we have been unable to develop a continuous assay.

3.2. Reconstitution for in vitro assays

Like other P450 enzymes, the reconstitution of recombinant P450 17A1 in a system containing NADPH-P450 reductase and phospholipids is required to obtain maximal catalytic activities. While some other P450 enzymes (e.g., P450 3A4) require particular phospholipids (Imaoka et al., 1992; Shaw et al., 1997), l-α-dilauroyl-sn-glycero-3-phosphocholine (DLPC) is typically used for P450 17A1 reactions. An accessory protein, cytochrome b5 (b5), is also required for lyase reactions, as described below.

It is well established that b5 stimulates P450 17A1 reactions (Katagiri et al., 1982; Katagiri et al., 1995). The 17α-hydroxylation activities can be observed without fortifying with b5 and are not enhanced much by the addition of b5, but P450 17A1 lyase activities are stimulated by b5 in a concentration-dependent manner (Fig. 2).

Fig. 2.

Fig. 2

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Stimulation of P450 17A1-catalyzed reactions by b5 (Kim et al., 2021). (A) 17α-hydroxylation activities of progesterone (●) and pregnenolone (■); (B) lyase activities of 17α-OH progesterone (●) and 17α-OH pregnenolone (■). Data represent means ± range of duplicate assays.

The enzyme reaction is initiated by adding an NADPH-generating system consisting of NADP+, glucose 6-phosphate, and yeast glucose 6-phosphate dehydrogenase, which is substituted for NADPH (Guengerich, 2014). A general protocol for the recombinant human P450 17A1 enzyme incubations are follows:

3.3. Materials

  • Recombinant human P450 17A1, expressed and purified as described above

  • Recombinant rat NADPH-P450 reductase (Hanna et al., 1998)

  • Recombinant human b5 (Guengerich, 2005)

  • 1.0 mg/mL DLPC (in H2O), freshly sonicated before use

  • NADPH-generating system. Checking the generating system according to the reported procedure before the experiment is recommended (ΔA340 increase) (Guengerich, 2014).

  • 1 M potassium phosphate buffer, pH 7.4

3.4. Enzyme incubations

  1. In a glass test tube, mix P450 17A1 (0.1–0.2 μM for 17α-hydroxylation, 0.2–0.5 μM for lyase reaction), NADPH-P450 reductase (2- to 10-× molecular ratio in excess of P450 17A1), b5 (1- to 10-× molecular ratio to P450 17A1, if needed), and 30 μM DLPC. Let the mixture stand on ice for 10 min

  2. Add 1 M potassium phosphate buffer (pH 7.4, final 50–100 mM), H2O, and substrate solution (generally prepared in CH3OH or CH3CN) in this order. Keep the concentration of organic solvents ≤ 1% (v/v).

  3. Divide the mixture into the desired number of reactions (500 μL final volume).

  4. Pre-incubate the mixture for 5 min at 37 °C.

  5. Initiate the reaction with the NAPDPH-generating system (0.5 mM NADP+, 10 mM glucose 6-phosphate, 2 μg/mL glucose 6-phosphate dehydrogenase) to initiate the reaction. The total volume will be 500 μL.

  6. Incubate for 5 min (for 17α-hydroxylation) or 10 min (for lyase reaction) at 37 °C, with gentle shaking.

  7. Quench the reaction by addition of 2.0 mL of organic solvent (ethyl acetate or CH2Cl2). Vortex vigorously to stop the enzymes.

  8. Transfer the majority of the organic layer (measured, e.g. 0.8 out of 1.0 mL) into a separate glass test tube and dry under a stream of N2 gas.

  9. For Δ4 steroid reactions, dissolve the residue in 100 μL of a CH3CN/H2O (1/1, v/v) mixture and analyze by HPLC as described below. For Δ5 steroid reactions, convert them into Δ4 steroids with cholesterol oxidase (Gonzalez & Guengerich, 2017; Kim et al., 2021) or derivatize them with dansylhydrazine, as described later.

4. Assay methods

4.1. Gas chromatography (GC)

Although some steroids can be analyzed by GC, in most cases derivatization is employed for GC and GC–MS analysis (Knapp, 1979). Procedures for preliminary separation of steroids by TLC and formation of silyl ethers has been used for analyzing these steroids (Kirschner & Lipsett, 1963), using a simple argon electron capture detector (Vandenheuvel et al., 1962). A more modern approach involves the use of a mass spectrometer as a detector and heavy isotope internal standards, but GC is a rather laborious process and has been largely replaced by HPLC methods.

4.2. Thin-layer chromatography (TLC)

TLC has been used to assay all the major P450 17A1 activities but requires the use of radioactive substrates (14C or 3H). The procedure involves extractions of the products with CH2Cl2, spotting on TLC, development with solvents, and detection/quantitation of the products. Typical solvent systems for TLC development are mixtures of CHCl3/ethyl acetate (Zuber et al., 1985; Zuber et al., 1986) and CH2Cl2/ethyl acetate/CH3OH (Zhou et al., 2005; Zhou et al., 2007). The general of migration (lowest Rf to highest) in progesterone > androstenedione > 17α-OH progesterone and pregnenolone > DHEA > 17α-OH pregnenolone. (Zhou et al., 2007; Zuber et al., 1986) Quantitation of radioactive products can be done by (i) autoradiography, (ii) scanning, or (iii) scraping and liquid scintillation counting.

The methods are sound but are problematic in the expense and inconvenience of radioisotopes and the time involved in manually extracting samples, spotting, and measuring radioactivity. Also, radioactive 17α-OH pregnenolone is not generally commercially available (it can be synthesized from radiolabeled pregnenolone by P450 17A1 in the absence of b5).

4.3. Liquid chromatography (HPLC, UPLC)

The presence of chromophores in the steroid nucleus—such as the (α,β)-unsaturated carbonyl of the Δ4-3-keto-steroids—permits their analysis via liquid chromatography coupled to ultraviolet (245 nm) detection (LC–UV) (Fig. 3). UV sensitivity, however, is often limited to steroid concentrations above %0.1–0.5 μM (~1–5 pmol) for the Δ4 steroids and is not suitable for Δ5 steroids, which lack strong chromophores. Consequently, the detection of Δ5 steroids requires both creativity in assay designs, sample analysis, and more sensitive instrumentation.

Fig. 3.

Fig. 3

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UPLC-UV of products of progesterone oxidation. Progesterone (Prog) and the products 17α-hydroxyprogesterone (17α-Prog) and androstenedione (Andro) are marked. The minor products 16α-progesterone, algesterone (16α,17α-dihydroxyprogesterone), and 21-hydroxyprogesterone are eluted earlier (Gonzalez & Guengerich, 2017; Yoshimoto et al., 2016).

4.3.1. Δ4 Steroids

The origin of the Δ4 steroid series is the oxidation of pregnenolone, the first oxidative product of cholesterol metabolism. This reaction—facilitated by a Δ5, Δ4 isomerase (3β-hydroxysteroid dehydrogenase)—proceeds via oxidation of the C3 hydroxyl group to a carbonyl, and subsequent migration of the C5–C6 double bond to C4–C5. The net product is the installation of an (α,β)-unsaturated carbonyl group across C3–C5, characteristic of the Δ4 steroids. This moiety is often the basis of analysis for many Δ4 steroids due to its strong UV character and corresponding sensitivity (~1–5 pmol) on most modern LC–UV systems.

4.3.1.1. HPLC

One common analytical method involves the use of radiolabeled (3H or 14C) steroids in enzyme incubations, followed by flow-counting to detect the radiolabel retained in the metabolites. Depending on the specific radioactivity (mCi/mmol) of the labeled material, this method can offer increased sensitivity for the Δ4 steroids, although price poses a potential barrier to its implementation. General protocols for the analysis of Δ4 steroids based on UV absorbance and radiolabel retention are provided below.

4.3.1.1.1. UPLC-UV.

The ε240 of Δ4 3-keto-steroids is ~16,000 M−1 cm−1 (Ehrenstein & Stevens, 1941), which allows for detection of all products that retain the conjugated double bonds of the A ring (Fig. 3). Simple H2O-CH3OH gradients are useful for elution (Gonzalez & Guengerich, 2017; Yoshimoto et al., 2016). A general protocol for the detection of Δ4 steroids on the basis of UV absorbance is as follows (Guengerich, McCarty, Chapman, et al., 2021):

  1. Extract the products of a quenched reaction with CH2Cl2 (2 mL), transfer an aliquot (1.6 mL) to fresh vials, and dry under a steady stream of N2 gas.

  2. Resuspend dried residue in a CH3CN/sodium acetate mixture (25 mM, pH 3.7) (1:1, v/v) and transfer to vials for UPLC analysis.

  3. Note: Best performance will be observed when the final (injected) concentration of Δ4 steroid is above 0.1–0.5 μM, or the injected amount is at least 1–5 pmol.

  4. LC conditions: Hold samples at a low temperature (4 °C) prior to analysis, and inject (15 μL) on a Waters Acquity UPLC system (held at 25 °C) using a 2.1 mm × 100 mm Acquity BEH octadecylsilane (C18) column (1.7 μm). Separate analytes using an isocratic mobile phase of 25 mM aqueous ammonium acetate buffer (pH 3.7) and CH3CN (4:6, v/v) at a flow rate of 0.2 mL/min. Detect analytes using a Waters Acquity photodiode array system set at 240 nm.

  5. Data analysis: Absolute quantitation can be achieved by comparison of peak integrations to those of an authentic standard, or by calculation of percent conversion of substrate to product (on the basis of peak area ratios).

4.3.1.1.2. Radio-HPLC.

A general protocol for the detection of Δ4-3-keto-steroids on the basis of radiolabel retention is as follows (Guengerich, McCarty, Chapman, et al. 2021):

  1. Note: Analytical sensitivity may be optimal when the activity of the radiolabel is greater than ~ 100 mCi/mmol for 3H or ~ 50 mCi/mmol for 14C (Gonzalez & Guengerich, 2017; Guengerich, McCarty, Chapman, et al., 2021; Hargrove et al., 2020; Reddish & Guengerich, 2019).

  2. Extract the products of a quenched reaction with CH2Cl2 (2 mL), transfer an aliquot (1.6 mL) to fresh vials, and dry under a stream of N2 gas.

  3. Redissolve the dried residue in mobile phase (50% aqueous CH3CN (v/v), 125 μL) and transfer the solution to vials for HPLC analysis.

  4. HPLC conditions: Inject samples (100 μL, 4 °C) on an HPLC instrument (Agilent 1100) using a Beckman Ultrasphere 4.6 mm × 250 mm octadecylsilane column (C18) (5 μm) with a gradient of an aqueous 0.1% HCO2H solution (w/v) and CH3CN at a flow rate of 1 mL/min as follows: 0 min, 60% CH3CN; 4 min, 60% CH3CN; 10 min, 20% CH3CN; 11 min, 20% CH3CN; 12 min, 60% CH3CN; 14 min, 60% CH3CN (all v/v).

  5. Flow-counting conditions: Effluent from the C18 column should be mixed with a stream of scintillation cocktail according to the manufacturer’s instructions, however a ratio of 1:2 or 1:3 (v/v) is generally standard. Detect radioactivity with a β-RAM Model 5 system (IN/US, LabLogic, Tampa, FL).

  6. Data analysis: Absolute quantitation can be achieved by comparison of peak integrations to those of an authentic standard, or by calculation of percent conversion of substrate to product (on the basis of peak area ratios).

4.3.1.1.3. LC–MS.

Another approach is the use of a mass spectrometer as a detector. Atmospheric pressure chemical ionization (APCI) has advantages in the analysis of neutral steroids, in general, but positive ion electrospray can be utilized with the addition of 0.1% HCO2H (v/v) to the solvents and a multiple reaction mode (MRM) with the following transitions: progesterone (m/z 316.2 > 96.9), 17α-OH progesterone (m/z 331.2 > 96.9), androstenedione (m/z 287.2 > 96.9), pregnenolone (m/z 317.2 > 90.9), 17α-OH pregnenolone (m/z 297.1 > 105), and DHEA (m/z 271.2 > 105.0) (Lee et al., 2023).

4.3.1.1.4. LC–MS of Δ5 steroids converted to Δ4 steroids.

Another option is to extract the products with CH2Cl2 or another (water-immiscible) solvent, transfer the organic phase and dry, and then incubate with cholesterol oxidase (which has O2 as a co-substrate and also forms H2O2), and then analyze the products as Δ4 steroids with UV detection (vide supra) (Gonzalez & Guengerich, 2017).

4.3.1.1.5. LC–MS of derivatives.

Derivatization can be used to increase the sensitivity in LC analysis. Cheong et al. (2020) extracted products and used NH2OH to form oximes of the carbonyl products.

In our own experience, dansylation (Fig. 4) has provided a low cost and sensitive approach to analysis (Guengerich, McCarty, Chapman, et al., 2021). Although LC analysis can be done with an on-line fluorescence detector (excite 340 nm, emission 535 nm—or UV, for that matter), single-quadrupole positive ion electrospray mass spectrometry provides a >100-fold increase in sensitivity over fluorescence.

Fig. 4.

Fig. 4

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Structures of dansyl products. Note E and Z isomers (Guengerich, McCarty, Chapman et al., 2021).

4.3.2. Δ5 Steroids

The Δ5 steroids are those who retain a hydroxyl substituent at C3 and whose only point of unsaturation is the C5–C6 double bond. Due to the lack of strong chromophores in the Δ5 steroid frame (compared to the Δ4 3-ketosteroid series), conventional sample analysis with LC–UV is not a sensitive detection method. The detection of Δ5 steroids, then, will require both creativity in assay designs and sensitivity in instrumentation.

As discussed previously for the Δ4 steroids, sensitivity can be increased in some cases with the use of radiolabeled steroids in enzyme incubations, coupled with a flow-counting apparatus to detect their products. In fact, radiolabeled (3H or 14C) materials are commercially available for many of the Δ5 steroids and their intermediates. However, because reaching sub-pmol sensitivity with most flow-counting systems requires a high specific activity of the radiolabel, cost remains a potential barrier for this type of analysis. Not only can the price of the labeled chemical (per μCi) be expensive, but the cost of scintillation cocktail and flow-counting technology is often substantial as well.

Alternatives to flow-counting methods in steroid analysis have historically been found in the realm of chemical derivatization coupled to LC–MS detection. While LC–MS is a sensitive detection method for ionizable compounds, neutral steroids often have low ionization efficiency under most conditions. This issue can be addressed by chemical derivatization of the steroids and their metabolites to adduct an ionizable group to the steroid frame. In fact, the labile carbonyl groups of some steroids present a unique opportunity for chemical derivatization. For example, steroid ketones can be readily labeled with hydrazines under acidic conditions, and the resulting hydrazone products (positively charged at low pH conditions) are readily detected via electrospray ionization (ESI) LC–MS. Even a single-quadrupole instrument has been shown to reach sub-nM (<10 fmol) sensitivity when detecting derivatized dehydroepiandrosterone (Δ5 androgen). Thus, LC–MS detection of steroid derivatives is perhaps the most sensitive option for steroid analysis.

The sensitive detection of Δ5 steroids via radiolabel retention and chemical derivatization coupled with LC–MS will be described in the following section.

4.3.2.1. Radio-HPLC

A general protocol for the use of radiolabeled steroids in an enzyme incubation can be found under the assay conditions described for the Δ4 steroids. Activity of the radiolabel, extraction and analysis conditions are often identical for the Δ5 steroids.

4.3.2.2. Dansyl-NHNH2/LC–MS

Dansylhydrazine is an inexpensive and effective hydrazine compound that readily reacts with steroid ketones (Guengerich, McCarty, Chapman, et al., 2021). In the reaction of a Δ5 steroid with dansylhydrazine, the electro-philic carbonyl carbon (C20) is attacked by the nucleophilic primary amine, and subsequent loss of water generates a hydrazone derivative (Fig. 4). Once synthesized, the derivatives must be handled with care, as they are sensitive to light, heat, and acid-catalyzed hydrolysis. These derivatives are also fluorescent and are known to perform well (albeit less sensitively) using LC-fluorescence assays (see Section 4.3.1.1.5).

For ESI-MS detection, this method is perhaps most readily applicable to the analysis of the Δ5 steroids, as only the C20 ketone is available for derivatization. When applied to the Δ4 steroids, the additional carbonyl (C3) must also be considered as it readily derivatizes in the presence of dansylhydrazine, leading to multiple products (Figs. 4, 5). In this reaction, simultaneous derivatization of both carbonyls was found to be preferred, and four major products (E and Z isomers at two carbons) were observed. A general protocol for the derivatization of steroids with dansylhydrazine is as follows:

Fig. 5.

Fig. 5

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UPLC–MS detection of dansylated oxidation products (Guengerich, McCarty, Chapman, et al., 2021). Residual substrates are also shown. (A) 17α-OH pregnenolone; (B) DHEA; (C) progesterone; (D) 17α-OH progesterone. The first set of doublets (tR 5.5–6.0 min) in the 17-OH progesterone chromatogram (Part D) corresponds to 16α-OH progesterone and the second set (tR 6.2–6.6 min) corresponds to 17α-OH progesterone. The chromatography conditions differ from those used in Fig. S2. In Part B, the incubation was done with 20 nM P450 17A1 for 5 min at 37 °C to obtain the product trace shown. In Part D, the incubation was done with 250 nM P450 17A1 for 3 min at 23 °C to obtain the product trace shown.

  1. Extract the products of a quenched reaction with CH2Cl2 (2 mL), transfer aliquots (1.6 mL) to fresh vials, and dry under a stream of N2 gas.

  2. Resuspend the dried residue in a mixture of CF3CO2H (7 mM, prepared as a 0.1% v/v solution in C2H5OH) and dansylhydrazine (10 mM, added from a 25 mM solution prepared in CH3OH) in amber glass vials. Vortex lightly for mixing.

  3. Cap the vials and allow the mixture to incubate overnight (16 h) in the dark at room temperature (23 °C). Shaking of the samples is not required.

  4. Quench the excess dansylhydrazine with acetone (50 mM, prepared as a 1 M solution in CH3OH) and incubate (23 °C) for 30 min in the dark (a closed drawer will suffice).

  5. Neutralize the reaction with NaOH (100 mM) and extract the products of the derivatization into CH2Cl2 (1 mL).

  6. Remove a measured aliquot (0.8 mL) of the organic (lower) layer and transfer to fresh amber vials. Bring to dryness under a steady stream of N2 gas.

  7. Dissolve the dried residue in a mixture (100 μL) of mobile phase (1:1 A:B, A= 0.1% HCO2H (aqueous), B=CH3CN). Transfer solution to LC autosampler vials for analysis.

  8. LC conditions: Inject samples (10 μL) on a Waters Acquity UPLC (40 °C) using a 2.1 mm × 50 mm Acquity BEH C18 column (1.7 μm) and separate analytes at a flow rate of 0.2 mL/min using a gradient of solutions of (A) 0.1% HCO2H (aqueous) and B=CH3CN as follows (all v/v): 0 min, 60% A; 0.5 min, 60% A; 8 min, 0% A; 8.5 min, 5% A; 9 min, 0% A; 9.1 min, 60% A; 10 min, 60% A.

  9. MS conditions: Detect analytes of interest with an on-line single quadrupole mass spectrometer (Waters QDa Detector, positive ion mode) using a cone voltage of 15 V, a sampling frequency of 10.0 Hz, and scanning from m/z 150–800. Process data using MassLynx software (Waters). Compare peak area integrations to those of a derivatized standard curve for absolute quantitation.

  10. Data analysis: In the MS chromatogram, a large peak at m/z 306 will correspond to the derivatized quenching reagent (acetone). Absolute product quantitation can be achieved by comparison of peak integrations to those of an authentic standard, or by calculation of percent conversion of substrate to product (on the basis of peak area ratios).

5. Issues in the P450 17A1 field

5.1. Inhibitors

P450 17A1 is a drug target in androgen-stimulated maladies (e.g., prostate cancer, polycystic ovary disease), and not surprisingly numerous inhibitors have been developed with potential as drugs. The list of inhibitors includes the azoles ketoconazole and clotrimazole and three other nitrogen heterocycles—abiraterone, orteronel, and seviteronel. Only abiraterone is in clinical use (as its 3-acetoxy prodrug, Zytiga®). Structures of human P450 17A1 with several of these inhibitors have been published by the Scott laboratory (DeVore & Scott, 2012; Fehl et al., 2018; Petrunak et al., 2017).

One of the problems in treating prostate cancer with inhibitors of P450 17A1 is that 17α-OH progesterone is needed for synthesis of glucocorticoids (e.g., cortisol). Ideally a drug could inhibit the lyase reaction but not the 17α-hydroxylation reaction (Bird & Abbott, 2016). Although some candidates have been claimed to be selective (Yamaoka et al., 2012), we and others have not observed any selectivity in favor of the lyase in our own assays (Burris-Hiday & Scott, 2021; Guengerich, McCarty, Chapman, et al., 2021; Petrunak et al., 2017).

The binding of inhibitors to P450 17A1, including all five mentioned above, is not a simple 1-step reaction. Detailed spectral kinetics analysis indicates that binding is at least a 3-step process (Guengerich, McCarty, Chapman, et al., 2021). However, full inhibition is achieved after the first step of binding of the inhibitor (Guengerich, McCarty, Chapman, et al., 2021) (in contrast to the case of P450 3A4, in which several steps are required for complete inhibition (Guengerich, McCarty, & Chapman, 2021)). Although the inhibition of P450 17A1 by abiraterone has been proposed to involve a slow, tight-binding mechanism (Cheong et al., 2020), we found no evidence for such a phenomenon, with complete inhibition occurring in a sub-second time scale (Guengerich, McCarty, Chapman, et al., 2021).

The difficulty of achieving selective inhibition of the lyase activity over 17α-hydroxylation, a desired pharmacological outcome (Bird & Abbott, 2016) can be seen in doing kinetic simulations (Guengerich et al., 2019), in that the substrates, products, and inhibitors all occupy the same space in the enzyme (Burris-Hiday & Scott, 2021; DeVore & Scott, 2012; Petrunak et al., 2017), although a very recent study has shown a peripheral binding site for some metabolites of the inhibitor abiraterone (Petrunak et al. 2023). An alternate approach might involve inhibiting the binding of b5 to P450 17A1, in that the lyase reaction is highly dependent on b5 but 17α-hydroxylation is not (Kim et al., 2021).

5.2. Species variation

Some major species differences exist. The mouse enzyme behaves in a similar way to the human, but its intrinsic activities (specificity constants, i.e. kcat/Km) are over an order of magnitude higher (Lee et al., 2023). In the rat, lyase activity is greater with Δ4 steroids (17α-OH progesterone) than Δ5 steroids (17α-OH pregnenolone) and lyase activities with both 17α-hydroxy steroids are much higher than with the human enzyme (Brock & Waterman, 1999). In teleost fish, P450 17A1 is rather similar to the human enzyme but a related second enzyme (P450 17A2) has very high 17α-hydroxylation activity but is devoid of lyase activity [although the active sites of the crystal structures are very similar (Pallan et al., 2015)].

5.3. Mechanism of b5 stimulation

b5 has been repeatedly shown to preferentially stimulate the lyase reactions, and the clinical observations with some single nucleotide variants of both b5 and P450 17A1 support this view in vivo (Auchus, 2017; Geller et al., 1999; Idkowiak et al., 2012; Kok et al., 2010). Although the Sligar laboratory has proposed that electron transfer is involved in the lyase stimulation (Duggal et al., 2016; Duggal et al., 2018), other laboratories have not observed this (Auchus et al., 1998; Guengerich et al., 2019; Lee-Robichaud et al., 1995), even in cells (Simonov et al., 2015). b5 has been shown to bind tightly to P450 17A1 (Kim et al., 2021), and the general consensus is that binding of b5 to P450 17A1 is an allosteric phenomenon that induces a change in the structure of P450 17A1 to enhance the lyase reactions (Auchus & Miller, 2015; Burris-Hiday & Scott, 2021).

5.4. Catalytic mechanism of the lyase reaction

The 17α-hydroxylation reaction is straightforward and generally agreed to utilize the usual Compound I (FeO3+) intermediate (Fig. 6). The lyase reaction is still more controversial. 18O2 incorporation experiments (incorporation into the product acetic acid) do not in themselves distinguish between Compound I (FeO3+) and Compound 0 (FeO2−) mechanisms. Arguments have been made for Compound I on the basis of solvent deuterium kinetic isotope effects (Gregory, Denisov, Grinkova, Khatri, & Sligar, 2013; Gregory, Mak, Sligar, & Kincaid, 2013), which are not definitive and have not been repeatable (Yoshimoto et al., 2016), and Raman spectroscopy of iron-oxygen complex intermediates (Gregory, Denisov et al., 2013; Gregory, Mak et al., 2013; Mak et al., 2018). However, the intermediates studies by Raman spectroscopy have never been examined for catalytic competence (i.e., product formation). A single-oxygen oxygen surrogate (iodosylbenzene) can support the lyase reaction, consistent with a role for Compound I, at least in the system used (Yoshimoto et al., 2016). A Compound I alternative has the FeO3+ entity attacking the 17α-OH group to form a peroxide (C-O-O-Fe), which can collapse to an oxetane and then the observed products (Gonzalez et al., 2018). In support of this mechanism, the addition of synthetic 17-OOH hydroperoxide steroids to P450 17A1 leads to the facile formation of androstenedione and DHEA in high yield (Gonzalez et al., 2018).

Fig. 6.

Fig. 6

Open in a new tab

P450 catalytic reaction cycle.

5.5. Ternary complex of redox partners vs ping-pong binding

The stimulation of P450 17A1 (lyase activity) by b5 presents an issue, in that several lines of investigation suggest that a certain region of P450 17A1 is involved in the interaction with both NADPH-P450 reductase (POR) and b5 (Geller et al., 1999; Peng et al., 2014). Some NMR evidence for direct competition of POR and b5 has been presented (Estrada et al., 2013, 2014, 2016), although the use of this technique requires high protein concentrations (>100 μM) that are probably orders of magnitude higher than the Kd values for binding (Kim et al., 2021). Such a ping-pong mechanism is also problematic in that POR would have to bind to P450 17A1 to move the iron to at least the FeO2− state and then leave an unstable P450 intermediate to allow room for b5 to bind, in each reaction cycle, in the time frame of stability of any unstable FeO complex. Our own fluorescence results, using modified fluorescent derivatives of b5 mutants, indicate that POR only partially restored the fluorescence attenuation due to binding b5 (Kim et al., 2021). Our interpretation of the results is that a ternary complex of P450 17A1, POR, and b5 exists, which we have supported with gel filtration studies (Kim et al., 2021).

Acknowledgments

We thank K. Trisler for assistance in preparation of the manuscript. Work in this area has been supported by National Institutes of Health Grant R01 GM118122. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This material is also based upon work supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. 1937963 (K.D.M.). Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.

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