Cytochrome P450 17A1 (CYP17A1, P450c17, EC 188.8.131.52) is a membrane-bound dual-function monooxygenase with a critical role in the synthesis of many human steroid hormones1
. CYP17A1 17α-hydroxylase activity is required for generation of glucocorticoids like cortisol, while its hydroxylase and 17,20-lyase activities are required for production of androgenic and estrogenic sex steroids (fig S1
). CYP17A1 is an important target for the treatment of breast and prostate cancers that proliferate in response to estrogens and androgens2,3
. In the absence of structural information, CYP17A1 inhibitors have been designed that are thought to bind the cytochrome P450 heme iron4
, but it has been difficult to rationalize or predict other structural features critical for effective, selective CYP17A1 inhibition. In addition, structural information is important to understand 17α-hydroxylase deficiencies and potentially polycystic ovary disease7
. We determined structures of human CYP17A1 bound to two clinically-relevant CYP17A1 inhibitors (fig. S2
). Abiraterone is the active form of a prodrug recently approved by the FDA for metastatic prostate cancer5,8
and under investigation for breast cancer9
. TOK-001 is currently in clinical trials for prostate cancer4
A truncated, His-tagged version of the human CYP17A1 protein was generated from a synthetic cDNA engineered to remove the single N-terminal transmembrane helix and expressed in E. coli
. Resulting CYP17A1 was membrane-bound, so was solubilized with detergent before purification. This CYP17A1 binds abiraterone () and TOK-001 (not shown) with absorbance decreases at 402 nm and increases at 424 nm, consistent with nitrogen binding to the heme iron (type II interaction) with Kd
values of <100 nM ( inset). Similar titrations with substrates progesterone () and pregnenolone (not shown) revealed absorbance decreases at 419 nm and increases at 385 nm, indicative of ligand displacing water from the heme (type I interaction). CYP17A1 binds pregnenolone (Kd
<100 nM, not shown) significantly more tightly than progesterone (Kd
229±14 nM; inset). In our hands, full-length enzyme10
had a similar kcat
and 3-fold higher Km
11.4±0.7 μM) compared to the truncated form (kcat
3.7±0.3 μM). IC50
values for abiraterone (201±1 nM) were lower than for TOK-001 (503.0±1.0 nM) (). Thus, truncated human CYP17A1 is a functional enzyme in terms of ligand binding, catalytic function, and inhibition.
Function of CYP17A1 and inhibition by clinical compounds
Both structures with abiraterone (2.6 Å) and TOK-001 (2.4 Å) demonstrate the characteristic cytochrome P450 fold () and have four very similar protein copies in each asymmetric unit (Table S1
). Consistent with spectral binding data, abiraterone and TOK-001 bind with the nitrogen of the C17 pyridine or benzimidazole, respectively, forming a coordinate covalent bond with heme iron (). The steroid nucleus of these inhibitors rise at a 60° angle above the heme plane, directed between the F and G helices (), and essentially overlap (). The unsubstituted α face packs flat against the I helix where G301, A302, and adjacent residues form a highly complementary hydrophobic planar surface (). The 3β-OH groups of abiraterone () and TOK-001 () hydrogen bond with N202 in the F helix (~2.6 Å and ~2.4 Å, respectively).
Although inhibitors occupy the majority of the enclosed active site, the void extends beyond these ligands in several directions. First, the active site wall nearest the inhibitor β face is not as complementary to the steroid nucleus as for the α face. The C18 and C19 methyl groups project toward a crevice between the B′ helix, the β4 loop, and the loop following the F helix (). Only three side chains of the cavity wall come within 4 Å of C18 or C19. The cavity wall facing the β face of abiraterone or TOK-001 is primarily lined with hydrophobic atoms of A105, S106, A113, F114, I206, L209, V236, and V482 (), but there are two notable exceptions. R239 and D298 extend from the G and I helices, respectively, to orient their basic and acidic termini toward C6. These two polar side chains flank a substantial extension of the active site void adjacent C6. Second, in the abiraterone structure there is additional volume available adjacent to the pyridine ring bordered by V366, A367, I371, and V483 (), which is occupied by benzimidazole in the TOK-001 structure (). Finally, the most substantial active site cavity extension is from the 3β-OH of the inhibitors over the top of helix I and along the underside of helices F and G. This cavity is mostly lined by hydrophobic residues (I198, L243, F300), but its “roof” is bordered by several polar F and G helix residues (Y201, N202, R239, ) that interact with, or are located near, waters in this region. The cavity containing TOK-001 is similar but slightly smaller over helix I ().
The single direct hydrogen bond between inhibitors and the protein is part of a larger hydrogen bonding network. In the abiraterone complex this network involves N202, E305, several conserved water molecules, R239, the backbone carbonyl of G297, and in some molecules Y201 (). While Y201 is not within hydrogen bonding distance to these waters for molecules A and B, the side chain rotates slightly toward abiraterone in molecules C and D to interact with one or both of the water molecules. TOK-001 has a very similar hydrogen bonding network (fig S3
). These interactions are strongly reminiscent of those conserved in the androgen, estrogen, glucocorticoid, mineralocorticoid, and progesterone receptors11
(). In each receptor, the 3β-OH or 3-keto of steroids binds within a deep receptor pocket and forms hydrogen bonds with an arginine, a glutamine/glutamate, and often a conserved water molecule. These interactions are critical for ligand recognition by hormone receptors12
and may also contribute to CYP17A1 selectivity for pregnenolone, progesterone, and their 17α-hydroxy derivatives. Notably, TOK-001 is both a CYP17A1 inhibitor and androgen receptor antagonist13
and the similarity of these binding modes is likely the reason for this dual mechanism of action.
Hydrogen bond network with abiraterone
CYP17A1 compared to the androgen receptor and CYP11A1
Orientations of the native CYP17A1 substrates are of substantial value in understanding the function of this enzyme. Pregnenolone and progesterone were docked into the CYP17A1/abiraterone structure modeled as the Fe(IV)=O (compound I) catalytic state. Progesterone maintained the N202 hydrogen bond. The distances from C17 and C16 to the catalytic oxygen were 3.7 Å and 3.9 Å, respectively, consistent with the observed 17α-OH (major) and 16α-OH (minor) progesterone metabolites. The pregnenolone C17 atom was 3.6 Å from the compound I oxygen and the 3β-OH hydrogen bonded to N202 (Fig S4
). However, the active site topology may be altered in the presence of substrates and this is an important area for further investigation.
CYP17A1 can be compared to three other P450 enzymes involved in steroidogenesis or cholesterol metabolism with reported steroid complex structures: CYP19A1 (aromatase)14
; CYP11A1 (cholesterol side-chain cleavage enzyme)15
; and CYP46A1, a cholesterol 24-hydroxylase16
. Although all four enzymes maintain the canonical cytochrome P450 fold, the other three orient steroids in the opposite direction from CYP17A1. All three have the steroid ligand positioned over the K-L loop directed towards theβ1 sheet as in CYP11A1 (), instead of oriented toward helices F and G.
Over 50 CYP17A1 mutations have been identified, most in patients with 17-hydroxylase deficiencies. The biochemical effects of many clinical mutations can be rationalized by examining the CYP17A1 structure (Fig S5
, Table S2
). Mutations R96W, R125Q, H373D/N, and R440H/C all alter residues that directly interact with the heme propionates and likely disrupt heme binding, consistent with complete loss of activity17–22
. Mutations E305G, R347H/C, R358Q, and R449A eliminate only the lyase activity of CYP17A123–25
. E305 hydrogen bonds to N202 in the active site, suggesting a role in substrate positioning, while the other residues are on the proximal face of the protein (fig. S5
), consistent with their proposed role in cytochrome b5
, which promotes lyase activity. Finally, reduction of the minor 16α-hydroxyprogesterone metabolite is reported for the artificial mutation A105L27
, consistent with its location in the active site facing the β face where the additional bulk may reduce the steroid movement within the active site.
Abiraterone and TOK-001 have several features which make them effective inhibitors of CYP17A1: 1) a heterocyclic nitrogen that coordinates to the heme iron, 2) a planar α face to pack against the I helix, and 3) hydrogen bonding interactions of 3β-OH with conserved polar residues in a hydrogen binding network. These structures provide a model for the binding of substrates and other inhibitors that is very different from binding orientations previously proposed by homology modeling and docking studies and from those demonstrated in other steroid-metabolizing cytochrome P450 enzymes. Perhaps most importantly, the cavity observed is not bilobed as predicted by many modeling studies28,29
. CYP17A1 interactions with these inhibitors are instead more reminiscent of steroid binding to steroid receptors, which may be the genesis of the TOK-001 dual mechanism of action. Thus, these structures contribute to a better understanding of the function and inhibition of CYP17A1 in a way that should substantially benefit the understanding of enzyme dysfunction in clinical disease and enable structure-based drug design of CYP17A1 inhibitors for treating hormone-responsive cancers, especially prostate cancer.