Structure-Activity Relationship of Antibody-Oligonucleotide Conjugates: Evaluating Bioconjugation Strategies for Antibody-Phosphorodiamidate Morpholino Oligomer Conjugates for Drug Development.

Antibody-oligonucleotide conjugates (AOCs) are promising treatments for Duchenne muscular dystrophy (DMD). They work via induction of exon skipping and restoration of dystrophin protein in skeletal and heart muscles. The structure-activity relationships (SARs) of AOCs comprising antibody-phosphorodiamidate morpholino oligomers (PMOs) depend on several aspects of their component parts. We evaluate the SAR of antimouse transferrin receptor 1 antibody (αmTfR1)-PMO conjugates: cleavable and noncleavable linkers, linker location on the PMO, and the impact of drug-to-antibody ratios (DARs) on plasma pharmacokinetics (PK), oligonucleotide delivery to tissues, and exon skipping. AOCs containing a stable linker with a DAR9.7 were the most effective PMO delivery vehicles in preclinical studies. We demonstrate that αmTfR1-PMO conjugates induce dystrophin protein restoration in the skeletal and heart muscles of mdx mice. Our results show that αmTfR1-PMO conjugates are a potentially effective approach for the treatment of DMD.

Targeted tissue delivery of RNA therapeutics using antibody-oligonucleotide conjugates (AOCs).

Although targeting TfR1 to deliver oligonucleotides to skeletal muscle has been demonstrated in rodents, effectiveness and pharmacokinetic/pharmacodynamic (PKPD) properties remained unknown in higher species. We developed antibody-oligonucleotide conjugates (AOCs) towards mice or monkeys utilizing anti-TfR1 monoclonal antibodies (αTfR1) conjugated to various classes of oligonucleotides (siRNA, ASOs and PMOs). αTfR1 AOCs delivered oligonucleotides to muscle tissue in both species. In mice, αTfR1 AOCs achieved a > 15-fold higher concentration to muscle tissue than unconjugated siRNA. A single dose of an αTfR1 conjugated to an siRNA against Ssb mRNA produced > 75% Ssb mRNA reduction in mice and monkeys, and mRNA silencing was greatest in skeletal and cardiac (striated) muscle with minimal to no activity in other major organs. In mice the EC50 for Ssb mRNA reduction in skeletal muscle was >75-fold less than in systemic tissues. Oligonucleotides conjugated to control antibodies or cholesterol produced no mRNA reduction or were 10-fold less potent, respectively. Tissue PKPD of AOCs demonstrated mRNA silencing activity primarily driven by receptor-mediated delivery in striated muscle for siRNA oligonucleotides. In mice, we show that AOC-mediated delivery is operable across various oligonucleotide modalities. AOC PKPD properties translated to higher species, providing promise for a new class of oligonucleotide therapeutics.

Maximizing ER-α Degradation Maximizes Activity in a Tamoxifen-Resistant Breast Cancer Model: Identification of GDC-0927.

The further optimization of ER-α degradation efficacy of a series of ER modulators by refining side-chain substitution led to efficacious selective estrogen receptor degraders (SERDs). A fluoromethyl azetidine group was found to be preferred and resulted in the identification of bis-phenol chromene 17ha. In a tamoxifen-resistant breast cancer xenograft model, 17ha (ER-α degradation efficacy = 97%) demonstrated tumor regression, together with robust reduction of intratumoral ER-α levels. However, despite superior oral exposure, 5a (ER-α degradation efficacy = 91%) had inferior activity. This result suggests that optimizing ER-α degradation efficacy leads to compounds with robust effects in a model of tamoxifen-resistant breast cancer. Compound 17ha (GDC-0927) was evaluated in clinical trials in women with metastatic estrogen receptor-positive breast cancer.

Selective estrogen receptor degraders with novel structural motifs induce regression in a tamoxifen-resistant breast cancer xenograft.

Potent estrogen receptor ligands typically contain a phenolic hydrogen-bond donor. The indazole of the selective estrogen receptor degrader (SERD) ARN-810 is believed to mimic this. Disclosed herein is the discovery of ARN-810 analogs which lack this hydrogen-bond donor. These SERDs induced tumor regression in a tamoxifen-resistant breast cancer xenograft, demonstrating that the indazole NH is not necessary for robust ER-modulation and anti-tumor activity.

Identification of an Orally Bioavailable Chromene-Based Selective Estrogen Receptor Degrader (SERD) That Demonstrates Robust Activity in a Model of Tamoxifen-Resistant Breast Cancer.

About 75% of breast cancers are estrogen receptor alpha (ER-α) positive, and women typically initially respond well to antihormonal therapies such as tamoxifen and aromatase inhibitors, but resistance often emerges. Fulvestrant is a steroid-based, selective estrogen receptor degrader (SERD) that both antagonizes and degrades ER-α and shows some activity in patients who have progressed on antihormonal agents. However, fulvestrant must be administered by intramuscular injections that limit its efficacy. We describe the optimization of ER-α degradation efficacy of a chromene series of ER modulators resulting in highly potent and efficacious SERDs such as 14n. When examined in a xenograft model of tamoxifen-resistant breast cancer, 14n (ER-α degradation efficacy = 91%) demonstrated robust activity, while, despite superior oral exposure, 15g (ER-α degradation efficacy = 82%) was essentially inactive. This result suggests that optimizing ER-α degradation efficacy in the MCF-7 cell line leads to compounds with robust effects in models of tamoxifen-resistant breast cancer derived from an MCF-7 background.

The selective estrogen receptor downregulator GDC-0810 is efficacious in diverse models of ER+ breast cancer.

ER-targeted therapeutics provide valuable treatment options for patients with ER+ breast cancer, however, current relapse and mortality rates emphasize the need for improved therapeutic strategies. The recent discovery of prevalent ESR1 mutations in relapsed tumors underscores a sustained reliance of advanced tumors on ERα signaling, and provides a strong rationale for continued targeting of ERα. Here we describe GDC-0810, a novel, non-steroidal, orally bioavailable selective ER downregulator (SERD), which was identified by prospectively optimizing ERα degradation, antagonism and pharmacokinetic properties. GDC-0810 induces a distinct ERα conformation, relative to that induced by currently approved therapeutics, suggesting a unique mechanism of action. GDC-0810 has robust in vitro and in vivo activity against a variety of human breast cancer cell lines and patient derived xenografts, including a tamoxifen-resistant model and those that harbor ERα mutations. GDC-0810 is currently being evaluated in Phase II clinical studies in women with ER+ breast cancer.

Optimization of an indazole series of selective estrogen receptor degraders: Tumor regression in a tamoxifen-resistant breast cancer xenograft.

Selective estrogen receptor degraders (SERDs) have shown promise for the treatment of ER+ breast cancer. Disclosed herein is the continued optimization of our indazole series of SERDs. Exploration of ER degradation and antagonism in vitro followed by in vivo antagonism and oral exposure culminated in the discovery of indazoles 47 and 56, which induce tumor regression in a tamoxifen-resistant breast cancer xenograft.

Identification of GDC-0810 (ARN-810), an Orally Bioavailable Selective Estrogen Receptor Degrader (SERD) that Demonstrates Robust Activity in Tamoxifen-Resistant Breast Cancer Xenografts.

Approximately 80% of breast cancers are estrogen receptor alpha (ER-α) positive, and although women typically initially respond well to antihormonal therapies such as tamoxifen and aromatase inhibitors, resistance often emerges. Although a variety of resistance mechanism may be at play in this state, there is evidence that in many cases the ER still plays a central role, including mutations in the ER leading to constitutively active receptor. Fulvestrant is a steroid-based, selective estrogen receptor degrader (SERD) that both antagonizes and degrades ER-α and is active in patients who have progressed on antihormonal agents. However, fulvestrant suffers from poor pharmaceutical properties and must be administered by intramuscular injections that limit the total amount of drug that can be administered and hence lead to the potential for incomplete receptor blockade. We describe the identification and characterization of a series of small-molecule, orally bioavailable SERDs which are potent antagonists and degraders of ER-α and in which the ER-α degrading properties were prospectively optimized. The lead compound 11l (GDC-0810 or ARN-810) demonstrates robust activity in models of tamoxifen-sensitive and tamoxifen-resistant breast cancer, and is currently in clinical trials in women with locally advanced or metastatic estrogen receptor-positive breast cancer.

A clinically relevant androgen receptor mutation confers resistance to second-generation antiandrogens enzalutamide and ARN-509.

UNLABELLED: Despite the impressive clinical activity of the second-generation antiandrogens enzalutamide and ARN-509 in patients with prostate cancer, acquired resistance invariably emerges. To identify the molecular mechanisms underlying acquired resistance, we developed and characterized cell lines resistant to ARN-509 and enzalutamide. In a subset of cell lines, ARN-509 and enzalutamide exhibit agonist activity due to a missense mutation (F876L) in the ligand-binding domain of the androgen receptor (AR). AR F876L is sufficient to confer resistance to ARN-509 and enzalutamide in in vitro and in vivo models of castration-resistant prostate cancer (CRPC). Importantly, the AR F876L mutant is detected in plasma DNA from ARN-509-treated patients with progressive CRPC. Thus, selective outgrowth of AR F876L is a clinically relevant mechanism of second-generation antiandrogen resistance that can potentially be targeted with next-generation antiandrogens. SIGNIFICANCE: A missense mutation in the ligand-binding domain of the androgen receptor F876L confers resistance to the second-generation antiandrogens enzalutamide and ARN-509 in preclinical models of AR function and prostate cancer and is detected in plasma DNA from ARN-509-treated patients with progressive disease. These results chart a new path for the discovery and development of next-generation antiandrogens that could be coupled with a blood-based companion diagnostic to guide treatment decisions.

ARN-509: a novel antiandrogen for prostate cancer treatment.

Continued reliance on the androgen receptor (AR) is now understood as a core mechanism in castration-resistant prostate cancer (CRPC), the most advanced form of this disease. While established and novel AR pathway-targeting agents display clinical efficacy in metastatic CRPC, dose-limiting side effects remain problematic for all current agents. In this study, we report the discovery and development of ARN-509, a competitive AR inhibitor that is fully antagonistic to AR overexpression, a common and important feature of CRPC. ARN-509 was optimized for inhibition of AR transcriptional activity and prostate cancer cell proliferation, pharmacokinetics, and in vivo efficacy. In contrast to bicalutamide, ARN-509 lacked significant agonist activity in preclinical models of CRPC. Moreover, ARN-509 lacked inducing activity for AR nuclear localization or DNA binding. In a clinically valid murine xenograft model of human CRPC, ARN-509 showed greater efficacy than MDV3100. Maximal therapeutic response in this model was achieved at 30 mg/kg/d of ARN-509, whereas the same response required 100 mg/kg/d of MDV3100 and higher steady-state plasma concentrations. Thus, ARN-509 exhibits characteristics predicting a higher therapeutic index with a greater potential to reach maximally efficacious doses in man than current AR antagonists. Our findings offer preclinical proof of principle for ARN-509 as a promising therapeutic in both castration-sensitive and castration-resistant forms of prostate cancer.

An F-domain introduced by alternative splicing regulates activity of the zebrafish thyroid hormone receptor alpha.

Thyroid hormones (THs) play an important role in vertebrate development; however, the underlying mechanisms of their actions are still poorly understood. Zebrafish (Danio rerio) is an emerging vertebrate model system to study the roles of THs during development. In general, the response to THs relies on closely related proteins and mechanisms across vertebrate species, however some species-specific differences exist. In contrast to mammals, zebrafish has two TRalpha genes (thraa, thrab). Moreover, the zebrafish thraa gene expresses a TRalpha isoform (TRalphaA1) that differs from other TRs by containing additional C-terminal amino acids. C-terminal extensions, called "F domains", are common in other members of the nuclear receptor superfamily and modulate the response of these receptors to hormones. Here we demonstrate that the F-domain constrains the transcriptional activity of zebrafish TRalpha by altering the selectivity of this receptor for certain coactivator binding motifs. We found that the F-domain of zebrafish TRalphaA1 is encoded on a separate exon whose inclusion is regulated by alternative splicing, indicating a regulatory role of the F-domain in vivo. Quantitative expression analyses revealed that TRalphaA1 is primarily expressed in reproductive organs whereas TRalphaB and the TRalphaA isoform that lacks the F-domain (TRalphaA1-2) appear to be ubiquitous. The relative expression levels of these TRalpha transcripts differ in a tissue-specific manner suggesting that zebrafish uses both alternative splicing and differential expression of TRalpha genes to diversify the cellular response to THs.

The glucocorticoid receptor represses cyclin D1 by targeting the Tcf-beta-catenin complex.

The ability of glucocorticoids (GCs) to regulate cell proliferation plays an important role in their therapeutic use. The canonical Wnt pathway, which promotes the proliferation of many cancers and differentiated tissues, is an emerging target for the actions of GCs, albeit existing links between these signaling pathways are indirect. By screening known Wnt target genes for their ability to respond differently to GCs in cells whose proliferation is either positively or negatively regulated by GCs, we identified c-myc, c-jun, and cyclin D1, which encode rate-limiting factors for G(1) progression of the cell cycle. Here we show that in U2OS/GR cells, which are growth-arrested by GCs, the glucocorticoid receptor (GR) represses cyclin D1 via Tcf-beta-catenin, the transcriptional effector of the canonical Wnt pathway. We demonstrate that GR can bind beta-catenin in vitro, suggesting that GC and Wnt signaling pathways are linked directly through their effectors. Down-regulation of beta-catenin by RNA interference impeded the expression of cyclin D1 but not of c-myc or c-jun and had no significant effect on the proliferation of U2OS/GR cells. Although these results revealed that beta-catenin and cyclin D1 are not essential for the regulation of U2OS/GR cell proliferation, considering the importance of the Wnt pathway for proliferation and differentiation of other cells, the repression of Tcf-beta-catenin activity by GR could open new possibilities for tissue-selective GC therapies.

Role of the N-terminal extension of the (betaalpha)8-barrel enzyme indole-3-glycerol phosphate synthase for its fold, stability, and catalytic activity.

Indole-3-glycerol phosphate synthase (IGPS) catalyzes the fifth step in the biosynthesis of tryptophan. It belongs to the large and versatile family of (betaalpha)(8)-barrel enzymes but has an unusual N-terminal extension of about 40 residues. Limited proteolysis with trypsin of IGPS from both Sulfolobus solfataricus (sIGPS) and Thermotoga maritima (tIGPS) removes about 25 N-terminal residues and one of the two extra helices contained therein. To assess the role of the extension, the N-terminally truncated variants sIGPSDelta(1-26) and tIGPSDelta(1-25) were produced recombinantly in Escherichia coli, purified, and characterized in comparison to the wild-type enzymes. Both sIGPSDelta(1-26) and tIGPSDelta(1-25) have unchanged oligomerization states and turnover numbers. In contrast, their Michaelis constants for the substrate 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate are increased, and their resistance toward unfolding induced by heat and guanidinium chloride is decreased. sIGPSDelta(1-26) was crystallized, and its X-ray structure was solved at 2.8 A resolution. The comparison with the known structure of sIGPS reveals small differences that account for its reduced substrate affinity and protein stability. The structure of the core of sIGPSDelta(1-26) is, however, unchanged compared to sIGPS, explaining its retained catalytic activity and consistent with the idea that it evolved from the same ancestor as the phosphoribosyl anthranilate isomerase and the alpha-subunit of tryptophan synthase. These (betaalpha)(8)-barrel enzymes catalyze the reactions preceding and following IGPS in tryptophan biosynthesis but lack an N-terminal extension.

Target-specific utilization of transcriptional regulatory surfaces by the glucocorticoid receptor.

The glucocorticoid receptor (GR) activates or represses transcription depending on the sequence and architecture of the glucocorticoid response elements in target genes and the availability and activity of interacting cofactors. Numerous GR cofactors have been identified, but they alone are insufficient to dictate the specificity of GR action. Furthermore, the role of different functional surfaces on the receptor itself in regulating its targets is unclear, due in part to the paucity of known target genes. Using DNA microarrays and real-time quantitative PCR, we identified genes transcriptionally activated by GR, in a translation-independent manner, in two human cell lines. We then assessed in U2OS osteosarcoma cells the consequences of individually disrupting three GR domains, the N-terminal activation function (AF) 1, the C-terminal AF2, or the dimer interface, on activation of these genes. We found that GR targets differed in their requirements for AF1 or AF2, and that the dimer interface was dispensable for activation of some genes in each class. Thus, in a single cell type, different GR surfaces were used in a gene-specific manner. These findings have strong implications for the nature of gene response element signaling, the composition and structure of regulatory complexes, and the mechanisms of context-specific transcriptional regulation.

Finding specificity within a conserved interaction site.

Recently developed approaches to generate drugs that regulate hormone-induced gene activation focus on modulating the interaction of nuclear receptors with coactivators. A study by Geistlinger and Guy demonstrates the feasibility of this approach and provides surprising evidence for specificity within the conserved nuclear receptor:coactivator interaction surface.

A C-terminal inhibitory domain controls the activity of p63 by an intramolecular mechanism.

The human genome is far smaller than originally estimated, and one explanation is that alternative splicing creates greater proteomic complexity than a simple count of open reading frames would suggest. The p53 homologue p63, for example, is a tetrameric transcription factor implicated in epithelial development and expressed as at least six isoforms with widely differing transactivation potential. In particular, p63alpha isoforms contain a 27-kDa C-terminal region that drastically reduces their activity and is of clear biological importance, since patients with deletions in this C terminus have phenotypes very similar to patients with mutations in the DNA-binding domain. We have identified a novel domain within this C terminus that is necessary and sufficient for transcriptional inhibition and which acts by binding to a region in the N-terminal transactivation domain of p63 homologous to the MDM2 binding site in p53. Based on this mechanism, we provide a model that explains the transactivation potential of homo- and heterotetramers composed of different p63 isoforms and their effect on p53.