Translational and Therapeutic Evaluation of RAS-GTP Inhibition by RMC-6236 in RAS-Driven Cancers.

RAS-driven cancers comprise up to 30% of human cancers. RMC-6236 is a RAS(ON) multi-selective noncovalent inhibitor of the active, GTP-bound state of both mutant and wild-type variants of canonical RAS isoforms with broad therapeutic potential for the aforementioned unmet medical need. RMC-6236 exhibited potent anticancer activity across RAS-addicted cell lines, particularly those harboring mutations at codon 12 of KRAS. Notably, oral administration of RMC-6236 was tolerated in vivo and drove profound tumor regressions across multiple tumor types in a mouse clinical trial with KRASG12X xenograft models. Translational PK/efficacy and PK/PD modeling predicted that daily doses of 100 mg and 300 mg would achieve tumor control and objective responses, respectively, in patients with RAS-driven tumors. Consistent with this, we describe here objective responses in two patients (at 300 mg daily) with advanced KRASG12X lung and pancreatic adenocarcinoma, respectively, demonstrating the initial activity of RMC-6236 in an ongoing phase I/Ib clinical trial (NCT05379985). SIGNIFICANCE: The discovery of RMC-6236 enables the first-ever therapeutic evaluation of targeted and concurrent inhibition of canonical mutant and wild-type RAS-GTP in RAS-driven cancers. We demonstrate that broad-spectrum RAS-GTP inhibition is tolerable at exposures that induce profound tumor regressions in preclinical models of, and in patients with, such tumors. This article is featured in Selected Articles from This Issue, p. 897.

Chemical remodeling of a cellular chaperone to target the active state of mutant KRAS.

The discovery of small-molecule inhibitors requires suitable binding pockets on protein surfaces. Proteins that lack this feature are considered undruggable and require innovative strategies for therapeutic targeting. KRAS is the most frequently activated oncogene in cancer, and the active state of mutant KRAS is such a recalcitrant target. We designed a natural product-inspired small molecule that remodels the surface of cyclophilin A (CYPA) to create a neomorphic interface with high affinity and selectivity for the active state of KRASG12C (in which glycine-12 is mutated to cysteine). The resulting CYPA:drug:KRASG12C tricomplex inactivated oncogenic signaling and led to tumor regressions in multiple human cancer models. This inhibitory strategy can be used to target additional KRAS mutants and other undruggable cancer drivers. Tricomplex inhibitors that selectively target active KRASG12C or multiple RAS mutants are in clinical trials now (NCT05462717 and NCT05379985).

Discovery of RMC-5552, a Selective Bi-Steric Inhibitor of mTORC1, for the Treatment of mTORC1-Activated Tumors.

Hyperactivation of mTOR kinase by mutations in the PI3K/mTOR pathway or by crosstalk with other mutant cancer drivers, such as RAS, is a feature of many tumors. Multiple allosteric inhibitors of mTORC1 and orthosteric dual inhibitors of mTORC1 and mTORC2 have been developed as anticancer drugs, but their clinical utility has been limited. To address these limitations, we have developed a novel class of "bi-steric inhibitors" that interact with both the orthosteric and the allosteric binding sites in order to deepen the inhibition of mTORC1 while also preserving selectivity for mTORC1 over mTORC2. In this report, we describe the discovery and preclinical profile of the development candidate RMC-5552 and the in vivo preclinical tool compound RMC-6272. We also present evidence that selective inhibition of mTORC1 in combination with covalent inhibition of KRASG12C shows increased antitumor activity in a preclinical model of KRASG12C mutant NSCLC that exhibits resistance to KRASG12C inhibitor monotherapy.

Selective inhibitors of mTORC1 activate 4EBP1 and suppress tumor growth.

The clinical benefits of pan-mTOR active-site inhibitors are limited by toxicity and relief of feedback inhibition of receptor expression. To address these limitations, we designed a series of compounds that selectively inhibit mTORC1 and not mTORC2. These 'bi-steric inhibitors' comprise a rapamycin-like core moiety covalently linked to an mTOR active-site inhibitor. Structural modification of these components modulated their affinities for their binding sites on mTOR and the selectivity of the bi-steric compound. mTORC1-selective compounds potently inhibited 4EBP1 phosphorylation and caused regressions of breast cancer xenografts. Inhibition of 4EBP1 phosphorylation was sufficient to block cancer cell growth and was necessary for maximal antitumor activity. At mTORC1-selective doses, these compounds do not alter glucose tolerance, nor do they relieve AKT-dependent feedback inhibition of HER3. Thus, in preclinical models, selective inhibitors of mTORC1 potently inhibit tumor growth while causing less toxicity and receptor reactivation as compared to pan-mTOR inhibitors.

Half-life extension of peptidic APJ agonists by N-terminal lipid conjugation.

Agonism of the endothelial receptor APJ (putative receptor protein related to AT1; AT1: angiotensin II receptor type 1) has the potential to ameliorate congestive heart failure by increasing cardiac output without inducing hypertrophy. Although the endogenous agonist, pyr-apelin-13 (1), has shown beneficial APJ-mediated inotropic effects in rats and humans, such effects are short-lived given its extremely short half-life. Here, we report the conjugation of 1 to a fatty acid, providing a lipidated peptide (2) with increased stability that retains inotropic activity in an anesthetized rat myocardial infarction (MI) model. We also report the preparation of a library of 15-mer APJ agonist peptide-lipid conjugates, including adipoyl-γGlu-OEG-OEG-hArg-r-Q-hArg-P-r-NMeLeuSHK-G-Oic-pIPhe-P-DBip-OH (17), a potent APJ agonist with high plasma protein binding and a half-life suitable for once-daily subcutaneous dosing in rats. A correlation between subcutaneous absorption rate and lipid length/type of these conjugates is also reported.

Mechanistic Studies of Cytochrome P450 3A4 Time-Dependent Inhibition Using Two Cysteine-Targeting Electrophiles.

Experiments designed to identify the mechanism of cytochrome P450 inactivation are critical to drug discovery. Small molecules irreversibly inhibit P450 enzymatic activity via two primary mechanisms: apoprotein adduct formation or heme modification. Understanding the interplay between chemical structures of reactive electrophiles and the impact on CYP3A4 structure and function can ultimately provide insights into drug design to minimize P450 inactivation. In a previous study, raloxifene and N-(1-pyrene) iodoacetamide (PIA) alkylated CYP3A4 in vitro; however, only raloxifene influenced enzyme activity. Here, two alkylating agents with cysteine selectivity, PIA and pyrene maleimide (PM), were used to investigate this apparent compound-dependent disconnect between CYP3A4 protein alkylation and activity loss. The compound's effect on 1) enzymatic activity, 2) carbon monoxide (CO) binding capacity, 3) intact heme content, and 4) protein conformation were measured. Results showed that PM had a large time-dependent loss of enzyme activity, whereas PIA did not. The differential effect on enzymatic activity between PM and PIA was mirrored in the CO binding data. Despite disruption of CO binding, neither compound affected the heme concentrations, inferring there was no destruction or alkylation of the heme. Lastly, differential scanning fluorescence showed PM-treated CYP3A4 caused a shift in the onset temperature required to induce protein aggregation, which was not observed for CYP3A4 treated with PIA. In conclusion, alkylation of CYP3A4 apoprotein can have a variable impact on catalytic activity, CO binding, and protein conformation that may be compound-dependent. These results highlight the need for careful interpretation of experimental results aimed at characterizing the nature of P450 enzyme inactivation. SIGNIFICANCE STATEMENT: Understanding the mechanism of CYP3A4 time-dependent inhibition is critical to drug discovery. In this study, we use two cysteine-targeting electrophiles to probe how subtle variation in inhibitor structure may impact the mechanism of CYP3A4 time-dependent inhibition and confound interpretation of traditional diagnostic experiments. Ultimately, this simplified system was used to reveal insights into CYP3A4 biochemical behavior. The insights may have implications that aid in understanding the susceptibility of CYP enzymes to the effects of electrophilic intermediates generated via bioactivation.

Rapid screening and characterization of glutathione-trapped reactive metabolites using a polarity switch-based approach on a high-resolution quadrupole orbitrap mass spectrometer.

Formation of reactive metabolites that are capable to react with macromolecules could contribute to drug-induced toxicity. As part of early drug screening strategy to support small molecule structure-activity relationship analysis, glutathione (GSH) trapping is commonly used for the detection of reactive metabolites. When trapped, the GSH conjugates can be characterized using mass spectrometry (MS)-based methods. In the present study, an efficient method was developed for rapid identification and characterization of GSH-trapped metabolites with a single run using a quadrupole orbitrap high-resolution mass spectrometer. The selective ion monitoring of m/z 272.0888, a characteristic product ion corresponding to deprotonated γ-glutamic-dehydroalanyl-glycine in the negative ionization mode, was applied as a survey scan leveraging all ion fragmentation mode using in-source collision-induced dissociation. Detection of the extracted product ions within 5.0 Δppm mass accuracy indicated the presence of putative GSH conjugates. Incorporation of fast polarity switching option and multiple data-dependent acquisition scans in a single cycle allowed the determination of accurate mass and multiple MS/MS spectra of GSH conjugates in both negative and positive ionization modes, which featured rich fragments for structural characterization. The effectiveness of this method was evaluated with four model compounds including acetaminophen, clozapine, diclofenac, and nefazodone in both liver microsome and cryopreserved hepatocyte incubations. Successful characterization of multiple GSH conjugates in each case validated this method. Overall, this approach provided a sensitive tool for rapid detection and characterization of GSH conjugates in vitro. Notably, this method could be suitable for high-throughput screening of reactive metabolites in the early drug discovery process. Graphical abstract Sensitive detection and characterization of glutatione conjugates using a high-resolution quadrupole orbitrap mass spectrometer.

In Vitro Metabolism of Oprozomib, an Oral Proteasome Inhibitor: Role of Epoxide Hydrolases and Cytochrome P450s.

Oprozomib is an oral proteasome inhibitor currently under investigation in patients with hematologic malignancies or solid tumors. Oprozomib elicits potent pharmacological actions by forming a covalent bond with the active site N-terminal threonine of the 20S proteasome. Oprozomib has a short half-life across preclinical species and in patients due to systemic clearance via metabolism. Potential for drug-drug interactions (DDIs) could alter the exposure of this potent therapeutic; therefore, a thorough investigation of pathways responsible for metabolism is required. In the present study, the major drug-metabolizing enzyme responsible for oprozomib metabolism was identified in vitro. A diol of oprozomib was found to be the predominant metabolite in human hepatocytes, which formed via direct epoxide hydrolysis. Using recombinant epoxide hydrolases (EHs) and selective EH inhibitors in liver microsomes, microsomal EH (mEH) but not soluble EH (sEH) was found to be responsible for oprozomib diol formation. Coincubation with 2-nonylsulfanyl-propionamide, a selective mEH inhibitor, resulted in a significant decrease in oprozomib disappearance (>80%) with concurrent complete blockage of diol formation in human hepatocytes. On the contrary, a selective sEH inhibitor did not affect oprozomib metabolism. Pretreatment of hepatocytes with the pan-cytochrome P450 (P450) inhibitor 1-aminobenzotriazole resulted in a modest reduction (∼20%) of oprozomib metabolism. These findings indicated that mEH plays a predominant role in oprozomib metabolism. Further studies may be warranted to determine whether drugs that are mEH inhibitors cause clinically significant DDIs with oprozomib. On the other hand, pharmacokinetics of oprozomib is unlikely to be affected by coadministered P450 and sEH inhibitors and/or inducers.

Development of a microphysiological model of human kidney proximal tubule function.

The kidney proximal tubule is the primary site in the nephron for excretion of waste products through a combination of active uptake and secretory processes and is also a primary target of drug-induced nephrotoxicity. Here, we describe the development and functional characterization of a 3-dimensional flow-directed human kidney proximal tubule microphysiological system. The system replicates the polarity of the proximal tubule, expresses appropriate marker proteins, exhibits biochemical and synthetic activities, as well as secretory and reabsorptive processes associated with proximal tubule function in vivo. This microphysiological system can serve as an ideal platform for ex vivo modeling of renal drug clearance and drug-induced nephrotoxicity. Additionally, this novel system can be used for preclinical screening of new chemical compounds prior to initiating human clinical trials.

Functional Comparison of Human Colonic Carcinoma Cell Lines and Primary Small Intestinal Epithelial Cells for Investigations of Intestinal Drug Permeability and First-Pass Metabolism.

To further the development of a model for simultaneously assessing intestinal absorption and first-pass metabolism in vitro, Caco-2, LS180, T84, and fetal human small intestinal epithelial cells (fSIECs) were cultured on permeable inserts, and the integrity of cell monolayers, CYP3A4 activity, and the inducibility of enzymes and transporters involved in intestinal drug disposition were measured. Caco-2, T84, and fSIECs all formed tight junctions, as assessed by immunofluorescence microscopy for zonula occludens-1, which was well organized into circumscribing strands in T84, Caco-2, and fSIECs but was diffuse in LS180 cells. The transepithelial electrical resistance value for LS180 monolayers was lower than that for Caco-2, T84, and fSIECs. In addition, the apical-to-basolateral permeability of the paracellular marker Lucifer yellow across LS180 monolayers was greater than in fSIECs, T84, and Caco-2 monolayers. The transcellular marker propranolol exhibited similar permeability across all cells. With regard to metabolic capacity, T84 and LS180 cells showed comparable basal midazolam hydroxylation activity and was inducible by rifampin and 1α,25(OH)2D3 in LS180 cells, but only marginally so in T84 cells. The basal CYP3A4 activity of fSIECs and Caco-2 cells was much lower and not inducible. Interestingly, some of the drug transporters expressed in LS180 and Caco-2 cells were induced by either 1α,25(OH)2D3 or rifampin or both, but effects were limited in the other two cell lines. These results suggest that none of the cell lines tested fully replicated the drug disposition properties of the small intestine and that the search for an ideal screening tool must continue.

Human UGT1A4 and UGT1A3 conjugate 25-hydroxyvitamin D3: metabolite structure, kinetics, inducibility, and interindividual variability.

25-Hydroxyvitamin D3 (25OHD3) is used as a clinical biomarker for assessment of vitamin D status. Blood levels of 25OHD3 represent a balance between its formation rate and clearance by several oxidative and conjugative processes. In the present study, the identity of human uridine 5'-diphosphoglucuronyltransferases (UGTs) capable of catalyzing the 25OHD3 glucuronidation reaction was investigated. Two isozymes, UGT1A4 and UGT1A3, were identified as the principal catalysts of 25OHD3 glucuronidation in human liver. Three 25OHD3 monoglucuronides (25OHD3-25-glucuronide, 25OHD3-3-glucuronide, and 5,6-trans-25OHD3-25-glucuronide) were generated by recombinant UGT1A4/UGT1A3, human liver microsomes, and human hepatocytes. The kinetics of 25OHD3 glucuronide formation in all systems tested conformed to the Michaelis-Menten model. An association between the UGT1A4*3 (Leu48Val) gene polymorphism with the rates of glucuronide formation was also investigated using human liver microsomes isolated from 80 genotyped livers. A variant allele dose effect was observed: the homozygous UGT1A4*3 livers (GG) had the highest glucuronidation activity, whereas the wild type (TT) had the lowest activity. Induction of UGT1A4 and UGT1A3 gene expression was also determined in human hepatocytes treated with pregnane X receptor/constitutive androstane receptor agonists, such as rifampin, carbamazepine, and phenobarbital. Although UGT mRNA levels were increased significantly by all of the known pregnane X receptor/constitutive androstane receptor agonists tested, rifampin, the most potent of the inducers, significantly induced total 25OHD3 glucuronide formation activity in human hepatocytes measured after 2, but not 4 and 24 hours, of incubation. Finally, the presence of 25OHD3-3-glucuronide in both human plasma and bile was confirmed, suggesting that the glucuronidation pathway might be physiologically relevant and contribute to vitamin D homeostasis in humans.

Enhancement of hepatic 4-hydroxylation of 25-hydroxyvitamin D3 through CYP3A4 induction in vitro and in vivo: implications for drug-induced osteomalacia.

Long-term therapy with certain drugs, especially cytochrome P450 (P450; CYP)-inducing agents, confers an increased risk of osteomalacia that is attributed to vitamin D deficiency. Human CYP24A1, CYP3A4, and CYP27B1 catalyze the inactivation and activation of vitamin D and have been implicated in the adverse drug response. In this study, the inducibility of these enzymes and monohydroxylation of 25-hydroxyvitamin D3 (25OHD3) were evaluated after exposure to P450-inducing drugs. With human hepatocytes, treatment with phenobarbital, hyperforin, carbamazepine, and rifampin significantly increased the levels of CYP3A4, but not CYP24A1 or CYP27B1 mRNA. In addition, rifampin pretreatment resulted in an 8-fold increase in formation of the major metabolite of 25OHD3, 4ß,25(OH)2D3. This inductive effect was blocked by the addition of 6',7'-dihydroxybergamottin, a selective CYP3A4 inhibitor. With human renal proximal tubular HK-2 cells, treatment with the same inducers did not alter CYP3A4, CYP24A1, or CYP27B1 expression. 24R,25(OH)2 D3 was the predominant monohydroxy metabolite produced from 25OHD3, but its formation was unaffected by the inducers. With healthy volunteers, the mean plasma concentration of 4ß,25(OH)2D3 was increased 60% (p < 0.01) after short-term rifampin administration. This was accompanied by a statistically significant reduction in plasma 1α,25(OH)2D3 (-10%; p = 0.03), and a nonsignificant change in 24R,25(OH)2D3 (-8%; p = 0.09) levels. Further analysis revealed a negative correlation between the increase in 4ß,25(OH)2D3 and decrease in 1α,25(OH)2D3 levels. Examination of the plasma monohydroxy metabolite/25OHD3 ratios indicated selective induction of the CYP3A4-dependent 4ß-hydroxylation pathway of 25OHD3 elimination. These results suggest that induction of hepatic CYP3A4 may be important in the etiology of drug-induced osteomalacia.

Innovations in preclinical biology: ex vivo engineering of a human kidney tissue microperfusion system.

Kidney disease is a public health problem that affects more than 20 million people in the US adult population, yet little is understood about the impact of kidney disease on drug disposition. Consequently there is a critical need to be able to model the human kidney and other organ systems, to improve our understanding of drug efficacy, safety, and toxicity, especially during drug development. The kidneys in general, and the proximal tubule specifically, play a central role in the elimination of xenobiotics. With recent advances in molecular investigation, considerable information has been gathered regarding the substrate profiles of the individual transporters expressed in the proximal tubule. However, we have little knowledge of how these transporters coupled with intracellular enzymes and influenced by metabolic pathways form an efficient secretory and reabsorptive mechanism in the renal tubule. Proximal tubular secretion and reabsorption of xenobiotics is critically dependent on interactions with peritubular capillaries and the interstitium. We plan to robustly model the human kidney tubule interstitium, utilizing an ex vivo three-dimensional modular microphysiological system with human kidney-derived cells. The microphysiological system should accurately reflect human physiology, be usable to predict renal handling of xenobiotics, and should assess mechanisms of kidney injury, and the biological response to injury, from endogenous and exogenous intoxicants.

Human PXR-mediated induction of intestinal CYP3A4 attenuates 1α,25-dihydroxyvitamin D3 function in human colon adenocarcinoma LS180 cells.

Oxidative catabolism of 1α,25-dihydroxyvitamin D(3) [1α,25(OH)(2)D(3)] is mediated by either CYP24A1 or CYP3A4. In this paper, we tested whether induction of CYP3A4 in the LS180 intestinal cell model enhances clearance of 1α,25(OH)(2)D(3) and blunts its hormonal effect on expression of the apical membrane calcium transport protein, TRPV6. Treatment with the hPXR agonist rifampin significantly increased CYP3A4 mRNA content and catalytic activity, but had no effect on CYP24A1 or TRPV6 mRNA content. Pre-treating cells with rifampin for 48h, prior to a 24h 1α,25(OH)(2)D(3) treatment phase, was associated with a subsequent 48% increase in the elimination of 1α,25(OH)(2)D(3) and a 35% reduction of peak TRPV6 mRNA. Introduction of the CYP3A4 inhibitor, 6',7'-dihydroxybergamottin, an active inhibitor in grapefruit juice, reversed the effects of rifampin on 1α,25(OH)(2)D(3) clearance and TRPV6 expression. Over-expression of hPXR in LS180 cells greatly enhanced the CYP3A4 responsiveness to rifampin pretreatment, and elicited a greater relative suppression of TRPV6 expression and an increase in 1α,25(OH)(2)D(3) disappearance rate, compared to vector expressed cells, following hormone administration. Together, these results suggest that induction of CYP3A4 in the intestinal epithelium by hPXR agonists can result in a greater metabolic clearance of 1α,25(OH)(2)D(3) and reduced effects of the hormone on the intestinal calcium absorption, which may contribute to an increased risk of drug-induced osteomalacia/osteoporosis in patients receiving chronic therapy with potent hPXR agonists. Moreover, ingestion of grapefruit juice in the at-risk patients could potentially prevent this adverse drug effect.

An inducible cytochrome P450 3A4-dependent vitamin D catabolic pathway.

Vitamin D(3) is critical for the regulation of calcium and phosphate homeostasis. In some individuals, mineral homeostasis can be disrupted by long-term therapy with certain antiepileptic drugs and the antimicrobial agent rifampin, resulting in drug-induced osteomalacia, which is attributed to vitamin D deficiency. We now report a novel CYP3A4-dependent pathway, the 4-hydroxylation of 25-hydroxyvitamin D(3) (25OHD(3)), the induction of which may contribute to drug-induced vitamin D deficiency. The metabolism of 25OHD(3) was fully characterized in vitro. CYP3A4 was the predominant source of 25OHD(3) hydroxylation by human liver microsomes, with the formation of 4ß,25-dihydroxyvitamin D(3) [4ß,25(OH)(2)D(3)] dominating (V(max)/K(m) = 0.85 ml · min(-1) · nmol enzyme(-1)). 4ß,25(OH)(2)D(3) was found in human plasma at concentrations comparable to that of 1α,25-dihydroxyvitamin D(3), and its formation rate in a panel of human liver microsomes was strongly correlated with CYP3A4 content and midazolam hydroxylation activity. Formation of 4ß,25(OH)(2)D(3) in primary human hepatocytes was induced by rifampin and inhibited by CYP3A4-specific inhibitors. Short-term treatment of healthy volunteers (n = 6) with rifampin selectively induced CYP3A4-dependent 4ß,25(OH)(2)D(3), but not CYP24A1-dependent 24R,25-dihydroxyvitamin D(3) formation, and altered systemic mineral homeostasis. Our results suggest that CYP3A4-dependent 25OHD(3) metabolism may play an important role in the regulation of vitamin D(3) in vivo and in the etiology of drug-induced osteomalacia.

Unexpected hormonal activity of a catechol equine estrogen metabolite reveals reversible glutathione conjugation.

4-Hydroxyequilenin (4-OHEN) is a major phase I metabolite of the equine estrogens present in widely prescribed hormone replacement formulations. 4-OHEN is autoxidized to an electrophilic o-quinone that has been shown to redox cycle, generating ROS, and to covalently modify proteins and DNA and thus potentially to act as a chemical carcinogen. To establish the ability of 4-OHEN to act as a hormonal carcinogen at the estrogen receptor (ER), estrogen responsive gene expression and proliferation were studied in ER(+) breast cancer cells. Recruitment by 4-OHEN of ER to estrogen responsive elements (ERE) of DNA in MCF-7 cells was also studied and observed. 4-OHEN was a potent estrogen, with additional weak activity associated with binding to the arylhydrocarbon receptor (AhR). The potency of 4-OHEN toward classical ERalpha mediated activity was unexpected given the reported rapid autoxidation and trapping of the resultant quinone by GSH. Addition of thiols to cell cultures did not attenuate the estrogenic activity of 4-OHEN, and preformed thiol conjugates added to cell incubations only marginally reduced ERE-luciferase induction. On reaction of the 4OHEN-GSH conjugate with NADPH, 4-OHEN was observed to be regenerated at a rate dependent upon NADPH concentration, indicating that intracellular nonenzymatic and enzymatic regeneration of 4-OHEN accounts for the observed estrogenic activity of 4-OHEN. 4-OHEN is therefore capable of inducing chemical and hormonal pathways that may contribute to estrogen-dependent carcinogenesis, and trapping by cellular thiols does not provide a mechanism of termination of these pathways.

Redox cycling of catechol estrogens generating apurinic/apyrimidinic sites and 8-oxo-deoxyguanosine via reactive oxygen species differentiates equine and human estrogens.

Metabolic activation of estrogens to catechols and further oxidation to highly reactive o-quinones generates DNA damage including apurinic/apyrimidinic (AP) sites. 4-Hydroxyequilenin (4-OHEN) is the major catechol metabolite of equine estrogens present in estrogen replacement formulations, known to cause DNA strand breaks, oxidized bases, and stable and depurinating adducts. However, the direct formation of AP sites by 4-OHEN has not been characterized. In the present study, the induction of AP sites in vitro by 4-OHEN and the endogenous catechol estrogen metabolite, 4-hydroxyestrone (4-OHE), was examined by an aldehyde reactive probe assay. Both 4-OHEN and 4-OHE can significantly enhance the levels of AP sites in calf thymus DNA in the presence of the redox cycling agents, copper ion and NADPH. The B-ring unsaturated catechol 4-OHEN induced AP sites without added copper, whereas 4-OHE required copper. AP sites were also generated much more rapidly by 4-OHEN. For both catechol estrogens, the levels of AP sites correlated linearly with 8-oxo-dG levels, implying that depuriniation resulted from reactive oxygen species (ROS) rather than depurination of estrogen-DNA adducts. ROS modulators such as catalase, which scavenges hydrogen peroxide and a Cu(I) chelator, blocked the formation of AP sites. In MCF-7 breast cancer cells, 4-OHEN significantly enhanced the formation of AP sites with added NADH. In contrast, no significant induction of AP sites was detected in 4-OHE-treated cells. The greater redox activity of the equine catechol estrogen produces rapid oxidative DNA damage via ROS, which is enhanced by redox cycling agents and interestingly by NADPH-dependent quinone oxidoreductase.

Selective estrogen receptor modulator delivery of quinone warheads to DNA triggering apoptosis in breast cancer cells.

Estrogen exposure is a risk factor for breast cancer, and estrogen oxidative metabolites have been implicated in chemical carcinogenesis. Oxidation of the catechol metabolite of estrone (4-OHE) and the beta-naphthohydroquinone metabolite of equilenin (4-OHEN) gives o-quinones that produce ROS and damage DNA by adduction and oxidation. To differentiate hormonal and chemical carcinogensis pathways in estrogen receptor positive ER(+) cells, catechol or beta-naphthohydroquinone warheads were conjugated to the selective estrogen receptor modulator (SERM) desmethylarzoxifene (DMA). ER binding was retained in the DMA conjugates; both were antiestrogens with submicromolar potency in mammary and endometrial cells. Cytotoxicity, apoptosis, and caspase-3/7 activation were compared in ER(+) and ER(-)MDA-MB-231 cells, and production of ROS was detected using a fluorescent reporter. Comparison was made to DMA, isolated warheads, and a DMA-mustard. Conjugation of warheads to DMA increased cytotoxicity accompanied by induction of apoptosis and activation of caspase-3/7. Activation of caspase-3/7, induction of apoptosis, and cytotoxicity were all increased significantly in ER(+) cells for the DMA conjugates. ROS production was localized in the nucleus for conjugates in ER(+) cells. Observations are compatible with beta-naphthohydroquinone and catechol groups being concentrated in the nucleus by ER binding, where oxidation and ROS production result, concomitant with caspase-dependent apoptosis. The results suggest that DNA damage induced by catechol estrogen metabolites can be amplified in ER(+) cells independent of hormonal activity. The novel conjugation of quinone warheads to an ER-targeting SERM gives ER-dependent, enhanced apoptosis in mammary cancer cells of potential application in cancer therapy.