Investigating a new C2-symmetric testosterone dimer and its dihydrotestosterone analog: Synthesis, antiproliferative activity on prostate cancer cell lines and interaction with CYP3A4.

The synthesis of a 17α-linked C2-symmetric testosterone dimer and its dihydrotestosterone analog is reported. The dimers were synthesized using a short five-step reaction sequence with 28% and 38% overall yield for the testosterone and dihydrotestosterone dimer, respectively. The dimerization reaction was achieved by an olefin metathesis reaction with 2nd generation Hoveyda-Grubbs catalyst. The dimers and their corresponding 17α-allyl precursors were tested for the antiproliferative activity on androgen-dependent (LNCaP) and androgen-independent (PC3) prostate cancer cell lines. The effects on cells were compared with that of the antiandrogen cyproterone acetate (CPA). The results showed that the dimers were active on both cell lines, with an increased activity towards androgen-dependent LNCaP cells. However, the testosterone dimer (11) was fivefold more active than the dihydrotestosterone dimer (15), with an IC50 of 11.7 µM vs. 60.9 µM against LNCaP cells, respectively, and more than threefold more active than the reference drug CPA (IC50 of 40.7 µM). Likewise, studies on the interaction of new compounds with drug-metabolizing cytochrome P450 3A4 (CYP3A4) showed that 11 was a fourfold stronger inhibitor than 15 (IC50 of 3 µM and 12 µM, respectively). This suggests that changes in the chemical structure of sterol moieties and the manner of their linkage could largely affect both the antiproliferative activity of androgen dimers and their crossreactivity with CYP3A4.

Photosensitive Ru(II) Complexes as Inhibitors of the Major Human Drug Metabolizing Enzyme CYP3A4.

We report the synthesis and photochemical and biological characterization of the first selective and potent metal-based inhibitors of cytochrome P450 3A4 (CYP3A4), the major human drug metabolizing enzyme. Five Ru(II)-based derivatives were prepared from two analogs of the CYP3A4 inhibitor ritonavir, 4 and 6: [Ru(tpy)(L)(6)]Cl2 (tpy = 2,2':6',2″-terpyridine) with L = 6,6'-dimethyl-2,2'-bipyridine (Me2bpy; 8), dimethylbenzo[i]dipyrido[3,2-a:2',3'-c]phenazine (Me2dppn; 10) and 3,6-dimethyl-10,15-diphenylbenzo[i]dipyrido[3,2-a:2',3'-c]phenazine (Me2Ph2dppn; 11), [Ru(tpy)(Me2bpy)(4)]Cl2 (7) and [Ru(tpy)(Me2dppn)(4)]Cl2 (9). Photochemical release of 4 or 6 from 7-11 was demonstrated, and the spectrophotometric evaluation of 7 showed that it behaves similarly to free 4 (type II heme ligation) after irradiation with visible light but not in the dark. Unexpectedly, the intact Ru(II) complexes 7 and 8 were found to inhibit CYP3A4 potently and specifically through direct binding to the active site without heme ligation. Caged inhibitors 9-11 showed dual action properties by combining photoactivated dissociation of 4 or 6 with efficient 1O2 production. In prostate adenocarcinoma DU-145 cells, compound 9 had the best synergistic effect with vinblastine, the anticancer drug primarily metabolized by CYP3A4 in vivo. Thus, our study establishes a new paradigm in CYP inhibition using metalated complexes and suggests possible utilization of photoactive CYP3A4 inhibitory compounds in clinical applications, such as enhancement of therapeutic efficacy of anticancer drugs.

Innovative C2-symmetric testosterone and androstenedione dimers: Design, synthesis, biological evaluation on prostate cancer cell lines and binding study to recombinant CYP3A4.

The synthesis of two isomeric testosterone dimers and an androstenedione dimer is reported. The design takes advantage of an efficient transformation of testosterone leading to the synthesis of the key diene, 7α-(buta-1,3-dienyl)-4-androsten-17ß-ol-3-one, through an elimination reaction. It was found that in some instances the same reaction led to partial epimerization of the 17ß-hydroxyl group into the 17α-hydroxyl group. The specific orientation of the hydroxyl function was confirmed by NMR spectroscopy. Capitalizing on this unforeseen side reaction, several dimers were assembled using an olefin metathesis reaction with Hoveyda-Grubbs catalyst. This led to the formation of two isomeric testosterone dimers with 17α-OH or 17ß-OH (14α and 14ß) as well as an androstenedione dimer (14). The new dimers and their respective precursors were tested on androgen-dependent (LNCaP) and androgen independent (PC3 and DU145) prostate cancer cells. It was discovered that the most active dimer was made of the natural hormone testosterone (14ß) with an average IC50 of 13.3 µM. In LNCaP cells, 14ß was ∼5 times more active than the antiandrogen drug cyproterone acetate (IC50 of 12.0 µM vs. 59.6 µM, respectively). At low concentrations (0.25-0.5 µM), 14α and 14ß were able to completely inhibit LNCaP cell growth induced by testosterone or dihydrotestosterone. Furthermore, cross-reactivity of androgen-based dimers with sterol-metabolizing cytochrome P450 3A4 was explored and the results are disclosed herein.

Steroid bioconjugation to a CYP3A4 allosteric site and its effect on substrate binding and coupling efficiency.

Human cytochrome P450 3A4 (CYP3A4) is an important drug metabolizing enzyme involved in a number of drug-drug and food-drug interactions. As such, much effort has been devoted into investigating its mechanism of interaction with ligands. CYP3A4 has one of the highest levels of substrate promiscuity for an enzyme, and can even bind multiple ligands simultaneously. The location and orientation of these ligands depend on the chemical structure and stoichiometry, and are generally poorly understood. In the case of the steroid testosterone, up to three copies of the molecule can associate with the enzyme at once, likely two in the active site and one at a postulated allosteric site. Recently, we demonstrated that steroid bioconjugation at the allosteric site results in an increase in activity of CYP3A4 toward testosterone and 7-benzyloxy-4-trifluoromethylcoumarin oxidation. Here, using the established bioconjugation methodology, we show how steroid bioconjugation at the allosteric site affects the heme spin state, the binding affinity (KS) of CYP3A4 for testosterone, as well as the enzyme coupling efficiency.

Heme Binding Biguanides Target Cytochrome P450-Dependent Cancer Cell Mitochondria.

The mechanisms by which cancer cell-intrinsic CYP monooxygenases promote tumor progression are largely unknown. CYP3A4 was unexpectedly associated with breast cancer mitochondria and synthesized arachidonic acid (AA)-derived epoxyeicosatrienoic acids (EETs), which promoted the electron transport chain/respiration and inhibited AMPKα. CYP3A4 knockdown activated AMPKα, promoted autophagy, and prevented mammary tumor formation. The diabetes drug metformin inhibited CYP3A4-mediated EET biosynthesis and depleted cancer cell-intrinsic EETs. Metformin bound to the active-site heme of CYP3A4 in a co-crystal structure, establishing CYP3A4 as a biguanide target. Structure-based design led to discovery of N1-hexyl-N5-benzyl-biguanide (HBB), which bound to the CYP3A4 heme with higher affinity than metformin. HBB potently and specifically inhibited CYP3A4 AA epoxygenase activity. HBB also inhibited growth of established ER+ mammary tumors and suppressed intratumoral mTOR. CYP3A4 AA epoxygenase inhibition by biguanides thus demonstrates convergence between eicosanoid activity in mitochondria and biguanide action in cancer, opening a new avenue for cancer drug discovery.

High-Level Production and Properties of the Cysteine-Depleted Cytochrome P450 3A4.

Human drug-metabolizing cytochrome P450 3A4 (CYP3A4) is a dynamic enzyme with a large and highly malleable active site that can fit structurally diverse compounds. Despite extensive investigations, structure-function relationships and conformational dynamics in CYP3A4 are not fully understood. This study was undertaken to engineer a well-expressed and functionally active cysteine-depleted CYP3A4 that can be used in biochemical and biophysical studies. cDNA codon optimization and screening mutagenesis were utilized to boost the level of bacterial expression of CYP3A4 and identify the least harmful substitutions for all six non-heme-ligating cysteines. The C58A/C64M/C98A/C239T/C377A/C468S (Cys-less) mutant was found to be expressed as highly as the optimized wild-type (opt-WT) CYP3A4. The high-resolution X-ray structures of opt-WT and Cys-less CYP3A4 revealed that gene optimization leads to a different folding in the Phe108 and Phe189 regions and promotes binding of the active site glycerol that interlocks Ser119 and Arg212, critical for ligand association, and the hydrophobic cluster adjacent to Phe108. Crowding and decreased flexibility of the active site, as well as structural alterations observed at the C64M, C239T, and C468S mutational sites, might be responsible for the distinct ligand binding behavior of opt-WT and Cys-less CYP3A4. Nonetheless, the Cys-less mutant could be used for structure-function investigations because it orients bromoergocryptine and ritonavir (a high-affinity substrate and a high-potency inhibitor, respectively) like the WT and has a higher activity toward 7-benzyloxy(4-trifluoromethyl)coumarin.

Anion-Dependent Stimulation of CYP3A4 Monooxygenase.

We co-crystallized human cytochrome P450 3A4 (CYP3A4) with progesterone (PRG) under two different conditions, but the resulting complexes contained only one PRG molecule bound to the previously identified peripheral site. A novel feature in one of our structures is a citrate ion, originating from the crystallization solution. The citrate-binding site is located in an area where the N-terminus splits from the protein core and, thus, is suitable for the interaction with the anionic phospholipids of the microsomal membrane. We investigated how citrate affects the function of a soluble CYP3A4 monooxygenase system consisting of equimolar amounts of CYP3A4 and cytochrome P450 reductase (CPR). Citrate was found to affect the properties of both redox partners and stimulated their catalytic activities in a concentration-dependent manner via a complex mechanism. CYP3A4-substrate binding, reduction of CPR with NADPH, and interflavin and interprotein electron transfer were identified as citrate-sensitive steps. Comparative analysis of various negatively charged organic compounds indicated that, in addition to alterations caused by changes in ionic strength, anions modulate the properties of CYP3A4 and CPR through specific anion-protein interactions. Our results help to better understand previous observations and provide new mechanistic insights into CYP3A4 function.

Redox reactions of the FAD-containing apoptosis-inducing factor (AIF) with quinoidal xenobiotics: a mechanistic study.

Mitochondrial apoptosis-inducing factor (AIF) is a FAD-containing protein that under certain conditions translocates to the nucleus and causes a programmed cell death, apoptosis. The apoptogenic action of AIF is redox controlled as the NADH-reduced AIF dimer has lower affinity for DNA than the oxidized monomer. To gain further insights into the mechanism of AIF, we investigated its interaction with a series of quinone oxidants, including a number of anticancer quinones. Our data indicate that the NADH:quinone oxidoreduction catalyzed by AIF follows a "ping-pong" scheme, with the reductive half-reaction being rate-limiting and the FADH(-)-NAD(+) charge-transfer complex serving as an electron donor. AIF is equally reactive toward benzo- and naphthoquinones, but may discriminate structures with a higher number of aromatic rings. The reactivity of quinones is mainly defined by their one-electron reduction potential, whereas the size and nature of the substituents play a minor role. AIF is unlikely to significantly contribute to bioreductive activation of low-potential quinoidal anticancer quinones. However, high-potential quinones, e.g. a toxic natural compound naphthazarin, maintain AIF in the oxidized state when a significant excess of NADH is present. Thus, these compounds may prevent the accumulation of the reduced form of AIF in vivo, and enhance AIF-mediated apoptosis.

Redox-linked conformational dynamics in apoptosis-inducing factor.

Apoptosis-inducing factor (AIF) is a bifunctional mitochondrial flavoprotein critical for energy metabolism and induction of caspase-independent apoptosis, whose exact role in normal mitochondria remains unknown. Upon reduction with NADH, AIF undergoes dimerization and forms tight, long-lived FADH(2)-NAD charge-transfer complexes (CTC) that are proposed to be functionally important. To obtain a deeper insight into structure/function relations and redox mechanism of this vitally important protein, we determined the X-ray structures of oxidized and NADH-reduced forms of naturally folded recombinant murine AIF. Our structures reveal that CTC with the pyridine nucleotide is stabilized by (i) pi-stacking interactions between coplanar nicotinamide, isoalloxazine, and Phe309 rings; (ii) rearrangement of multiple aromatic residues in the C-terminal domain, likely serving as an electron delocalization site; and (iii) an extensive hydrogen-bonding network involving His453, a key residue that undergoes a conformational switch to directly interact with and optimally orient the nicotinamide for charge transfer. Via the His453-containing peptide, redox changes in the active site are transmitted to the surface, promoting AIF dimerization and restricting access to a primary nuclear localization signal through which the apoptogenic form is transported to the nucleus. Structural findings agree with biochemical data and support the hypothesis that both normal and apoptogenic functions of AIF are controlled by NADH.

Photoreduction of the active site of the metalloprotein putidaredoxin by synchrotron radiation.

X-ray damage to protein crystals is often assessed on the basis of the degradation of diffraction intensity, yet this measure is not sensitive to the rapid changes that occur at photosensitive groups such as the active sites of metalloproteins. Here, X-ray absorption spectroscopy is used to study the X-ray dose-dependent photoreduction of crystals of the [Fe(2)S(2)]-containing metalloprotein putidaredoxin. A dramatic decrease in the rate of photoreduction is observed in crystals cryocooled with liquid helium at 40 K compared with those cooled with liquid nitrogen at 110 K. Whereas structural changes consistent with cluster reduction occur in the active site of the crystal measured at 110 K, no such changes occur in the crystal measured at 40 K, even after an eightfold increase in dose. When the structural results from extended X-ray absorption fine-structure measurements are compared with those obtained by crystallography on this and similar proteins, it is apparent that X-ray-induced photoreduction has had an impact on the crystallographic data and subsequent structure solutions. These results strongly indicate the importance of using liquid-helium-based cooling for metalloprotein crystallography in order to avoid the subtle yet important changes that can take place at the metalloprotein active sites when liquid-nitrogen-based cooling is used. The study also illustrates the need for direct measurement of the redox states of the metals, through X-ray absorption spectroscopy, simultaneously with the crystallographic measurements.

Redox-dependent structural reorganization in putidaredoxin, a vertebrate-type [2Fe-2S] ferredoxin from Pseudomonas putida.

Putidaredoxin (Pdx), a vertebrate-type [2Fe-2S] ferredoxin from Pseudomonas putida, transfers electrons from NADH-putidaredoxin reductase to cytochrome P450cam. Pdx exhibits redox-dependent binding affinities for P450cam and is thought to play an effector role in the monooxygenase reaction catalyzed by this hemoprotein. To understand how the reduced form of Pdx is stabilized and how reduction of the [2Fe-2S] cluster affects molecular properties of the iron-sulfur protein, crystal structures of reduced C73S and C73S/C85S Pdx were solved to 1.45 angstroms and 1.84 angstroms resolution, respectively, and compared to the corresponding 2.0 angstroms and 2.03 angstroms X-ray models of the oxidized mutants. To prevent photoreduction, the latter models were determined using in-house radiation source and the X-ray dose received by Pdx crystals was significantly decreased. Structural analysis showed that in reduced Pdx the Cys45-Ala46 peptide bond flip initiates readjustment of hydrogen bonding interactions between the [2Fe-2S] cluster, the Sgamma atoms of the cysteinyl ligands, and the backbone amide nitrogen atoms that results in tightening of the Cys39-Cys48 metal cluster binding loop around the prosthetic group and shifting of the metal center toward the Cys45-Thr47 peptide. From the metal center binding loop, the redox changes are transmitted to the linked Ile32-Asp38 peptide triggering structural rearrangement between the Tyr33-Asp34, Ser7-Asp9 and Pro102-Asp103 fragments of Pdx. The newly established hydrogen bonding interactions between Ser7, Asp9, Tyr33, Asp34, and Pro102, in turn, not only stabilize the tightened conformation of the [2Fe-2S] cluster binding loop but also assist in formation of a specific structural patch on the surface of Pdx that can be recognized by P450cam. This redox-linked change in surface properties is likely to be responsible for different binding affinity of oxidized and reduced Pdx to the hemoprotein.

Crystal structure of putidaredoxin, the [2Fe-2S] component of the P450cam monooxygenase system from Pseudomonas putida.

Stability of the [2Fe-2S]-containing putidaredoxin (Pdx), the electron donor to cytochrome P450cam in Pseudomonas putida, was improved by mutating non-ligating cysteine residues, Cys73 and Cys85, to serine singly and in combination. The increasing order of stability is Cys73Ser/Cys85Ser>Cys73Ser>Cys85Ser>WT Pdx. Crystal structures of Cys73Ser/Cys85Ser and Cys73Ser mutants of Pdx, solved by single-wavelength anomalous dispersion phasing using the [2Fe-2S] iron atoms to 1.47 A and 1.65 A resolution, respectively, are nearly identical and very similar to those of bovine adrenodoxin (Adx) and Escherichia coli ferredoxin. However, unlike the Adx structure, no motion between the core and interaction domains of Pdx is observed. This higher conformational stability of Pdx might be due to the presence of a more extensive hydrogen bonding network at the interface between the two structural domains around the conserved His49. In particular, formation of a hydrogen bond between the side-chain of Tyr51 and the carbonyl oxygen atom of Glu77 and the presence of two well-ordered water molecules linking the interaction domain and the C-terminal peptide to the core of the molecule are unique to Pdx. The folding topology of the NMR model is similar to that of the X-ray structure of Pdx. The overall rmsd of Calpha positions between the two models is 1.59 A. The largest positional differences are observed for residues 18-21 and 33-37 in the loop regions and the C terminus. The latter two peptides display conformational heterogeneity in the crystal structures. Owing to flexibility, the aromatic ring of the C-terminal Trp106 can closely approach the side-chains of Asp38 and Thr47 (3.2-3.9 A) or move away and leave the active site solvent-exposed. Therefore, Trp106, previously shown to be important in the Pdr-to-Pdx and Pdx-to-P450cam electron transfer reactions is in a position to regulate and/or mediate electron transfer to or from the [2Fe-2S] center of Pdx.