Background: Structural information for substrate binding to human steroidogenic cytochrome P450 17A1 (CYP17A1) is unavailable. Results: Within a common overall orientation, different steroids adopt subtly different positions. Conclusion: Steric and hydrogen-bonding modulation of lateral/vertical orientation controls CYP17A1-mediated steroid oxidation. Significance: Understanding the CYP17A1 mechanism provides opportunities for better targeted drug design.

Human steroidogenic cytochrome P450 17A1 (CYP17A1) is a bifunctional enzyme that performs both hydroxylation and lyase reactions, with the latter required to generate androgens that fuel prostate cancer proliferation. The steroid abiraterone, the active form of the only CYP17A1 inhibitor approved by the Food and Drug Administration, binds the catalytic heme iron, nonselectively impeding both reactions and ultimately causing undesirable corticosteroid imbalance. Some nonsteroidal inhibitors reportedly inhibit the lyase reaction more than the preceding hydroxylase reaction, which would be clinically advantageous, but the mechanism is not understood. Thus, the nonsteroidal inhibitors seviteronel and orteronel and the steroidal inhibitors abiraterone and galeterone were compared with respect to their binding modes and hydroxylase versus lyase inhibition. Binding studies and X-ray structures of CYP17A1 with nonsteroidal inhibitors reveal coordination to the heme iron like the steroidal inhibitors. ()-seviteronel binds similarly to both observed CYP17A1 conformations. However, ()-orteronel and ()-orteronel bind to distinct CYP17A1 conformations that differ in a region implicated in ligand entry/exit and the presence of a peripheral ligand. To reconcile these binding modes with enzyme function, side-by-side enzymatic analysis was undertaken and revealed that neither the nonsteroidal seviteronel nor the ()-orteronel inhibitors demonstrated significant lyase selectivity, but the less potent ()-orteronel was 8- to 11-fold selective for lyase inhibition. While active-site iron coordination is consistent with competitive inhibition, conformational selection for binding of some inhibitors and the differential presence of a peripheral ligand molecule suggest the possibility of CYP17A1 functional modulation by features outside the active site.

Cytochrome P450 17A1 (CYP17A1) is an important target in the treatment of prostate cancer because it produces androgens required for tumour growth. The FDA has approved only one CYP17A1 inhibitor, abiraterone, which contains a steroidal scaffold similar to the endogenous CYP17A1 substrates. Abiraterone is structurally similar to the substrates of other cytochrome P450 enzymes involved in steroidogenesis, and interference can pose a liability in terms of side effects. Using nonsteroidal scaffolds is expected to enable the design of compounds that interact more selectively with CYP17A1. Therefore, we combined a structure-based virtual screening approach with density functional theory (DFT) calculations to suggest non-steroidal compounds selective for CYP17A1. In vitro assays demonstrated that two such compounds selectively inhibited CYP17A1 17α-hydroxylase and 17,20-lyase activities with IC 50 values in the nanomolar range, without affinity for the major drugmetabolizing CYP2D6 and CYP3A4 enzymes and CYP21A2, with the latter result confirmed in human H295R cells.Prostate cancer (PCa) is the second most common type of cancer in men and the fifth leading cause of death worldwide 1 . Several treatments have been developed against PCa, but drug resistance occurs rapidly, leading to a disease state known as castration-resistant prostate cancer (CRPC) 2,3 . In CRPC, androgens produced by the tumour and/or the adrenal gland drive disease progression. Thus, reduction or suppression of hormone levels in the cancer cells remains a key point in advanced stages of the disease.Cytochrome P450 17A1 (CYP17A1) is a monooxygenase involved in the synthesis of steroidal hormones. CYP17A1 converts pregnenolone to dehydroepiandrosterone and progesterone to androstenedione by two subsequent reactions, the 17α -hydroxylase and 17,20-lyase reactions (cf. Fig. 1). The hydroxylase reaction generates intermediates in the biosynthesis of glucocorticoids, while both hydroxylase and lyase reactions are required for biosynthesis of androgens and oestrogens 4 . CYP17A1 is therefore a pivotal target in the treatment of hormone-dependent tumours such as prostate cancer [5][6][7] .Several CYP17A1 inhibitors have been developed over the years, but only abiraterone (cf. Fig. 2) has been approved by the FDA for treating CRPC. Abiraterone consists of a steroidal scaffold with a pyridin-3-yl moiety in position 17 that inhibits CYP17A1 through coordination to the haem iron 8 . Oxygen binding to the haem iron is necessary for all CYP17A1 catalysis, so abiraterone binding is inhibitory. Together, the steroidal scaffold and the aromatic nitrogen-containing ring give abiraterone a promiscuous profile with affinity toward steroid receptors and other CYP enzymes, which likely contribute to the undesirable side effects observed in patients receiving abiraterone treatment 9 . Combinatorial synthesis programmes have been started by pharmaceutical companies to identify non-steroidal inhibitors and two such compounds, orteronel 10 and VT-464 11 , have been evaluate...

Edited by Norma AllewellThe transforming growth factor ␤ isoforms, TGF-␤1, -␤2, and -␤3, are small secreted homodimeric signaling proteins with essential roles in regulating the adaptive immune system and maintaining the extracellular matrix. However, dysregulation of the TGF-␤ pathway is responsible for promoting the progression of several human diseases, including cancer and fibrosis. Despite the known importance of TGF-␤s in promoting disease progression, no inhibitors have been approved for use in humans. Herein, we describe an engineered TGF-␤ monomer, lacking the heel helix, a structural motif essential for binding the TGF-␤ type I receptor (T␤RI) but dispensable for binding the other receptor required for TGF-␤ signaling, the TGF-␤ type II receptor (T␤RII), as an alternative therapeutic modality for blocking TGF-␤ signaling in humans. As shown through binding studies and crystallography, the engineered monomer retained the same overall structure of native TGF-␤ monomers and bound T␤RII in an identical manner. Cell-based luciferase assays showed that the engineered monomer functioned as a dominant negative to inhibit TGF-␤ signaling with a K i of 20 -70 nM. Investigation of the mechanism showed that the high affinity of the engineered monomer for T␤RII, coupled with its reduced ability to non-covalently dimerize and its inability to bind and recruit T␤RI, enabled it to bind endogenous T␤RII but prevented it from binding and recruiting T␤RI to form a signaling complex. Such engineered monomers provide a new avenue to probe and manipulate TGF-␤ signaling and may inform similar modifications of other TGF-␤ family members.The transforming growth factor ␤ isoforms, TGF-␤1, -␤2, and -␤3, are small secreted signaling proteins. Their overall structures are similar and consist of two cystine-knotted monomers tethered together by a single inter-chain disulfide bond (1). They coordinate wound healing, modulate immune cell function, maintain the extracellular matrix, and regulate epithelial and endothelial cell growth and differentiation (2). The TGF-␤s are synthesized as pre-pro-proteins, and after maturation, secretion, and release from their pro-domains (3), the mature homodimeric growth factors (GFs) 3 bind and bring together two single-pass transmembrane receptors, known as T␤RI and T␤RII, to form the signaling-competent T␤RI 2 -T␤RII 2 heterotetramer (4, 5). TGF-␤ GFs assemble T␤RI 2 -T␤RII 2 heterotetramer in a sequential manner, first by binding T␤RII followed by recruitment of T␤RI (6, 7). The stepwise assembly of T␤RII and T␤RI into a heterotetramer is driven by binding of T␤RI to a composite TGF-␤/T␤RII interface (Fig. 1A) (8, 9).The disruption or dysregulation of the TGF-␤ pathway is responsible for several human diseases. These include connec-

We pursued a structure-guided approach toward the development of improved dihydroorotate dehydrogenase (DHODH) inhibitors with the goal of forming new interactions between DHODH and the brequinar class of inhibitors. Two potential residues, T63 and Y356, suitable for novel H-bonding interactions, were identified in the brequinar-binding pocket. Analogues were designed to maintain the essential pharmacophore and form new electrostatic interactions through strategically positioned H-bond accepting groups. This effort led to the discovery of potent quinoline-based analogues 41 (DHODH IC = 9.71 ± 1.4 nM) and 43 (DHODH IC = 26.2 ± 1.8 nM). A cocrystal structure between 43 and DHODH depicts a novel water mediated H-bond interaction with T63. Additional optimization led to the 1,7-naphthyridine 46 (DHODH IC = 28.3 ± 3.3 nM) that forms a novel H-bond with Y356. Importantly, compound 41 possesses significant oral bioavailability ( F = 56%) and an elimination t = 2.78 h (PO dosing). In conclusion, the data supports further preclinical studies of our lead compounds toward selection of a candidate for early-stage clinical development.

Inhibition of embryonic ectoderm development (EED) is a new cancer therapeutic strategy. Herein, we report our discovery of EEDi-5285 as an exceptionally potent, efficacious, and orally active EED inhibitor. EEDi-5285 binds to the EED protein with an IC 50 value of 0.2 nM and inhibits cell growth with IC 50 values of 20 pM and 0.5 nM in the Pfeiffer and KARPAS422 lymphoma cell lines, respectively, carrying an EZH2 mutation. EEDi-5285 is approximately 100 times more potent than EED226 in binding to EED and >300 times more potent than EED226 in inhibition of cell growth in the KARPAS422 cell line. EEDi-5285 has excellent pharmacokinetics and achieves complete and durable tumor regression in the KARPAS422 xenograft model in mice with oral administration. The cocrystal structure of EEDi-5285 in a complex with EED defines the precise structural basis for their high binding affinity. EEDi-5285 is the most potent and efficacious EED inhibitor reported to date.

Cytochrome P450 17A1 (CYP17A1) operates at the core of human steroidogenesis, directing precursors into mineralocorticoids, glucocorticoids, or sex steroids. Although the 17α–hydroxylase and 17,20-lyase activities of this dual function enzyme have been investigated extensively, until recently no CYP17A1 structures were available to inform our understanding. Structures of CYP17A1 with a range of steroidal inhibitors and substrates are now available. This review relates functional knowledge of this enzyme to structural features defining the selective differentiation between its various substrates. While both hydroxylase and lyase substrates have similar orientations with respect to the heme, subtle differences in hydrogen bonding between CYP17A1 and the C3 substituent at the opposite end of ligands appear to correlate with differential substrate utilization and product formation. Complementary structural information from solution NMR supports cytochrome b5 allosteric modulation of the lyase reaction, implicating regions involved in ligand access to the otherwise buried active site.

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