Full Antagonist Research Articles

Abstract 3406: GDC-9545: A pure antiestrogen clinical candidate that immobilizes the estrogen receptor and profoundly alters chromatin accessibility in vivo

Abstract Estrogen Receptor-positive (ER+) breast cancer is a significant unmet medical need. Tumors expressing ER exhibit evidence of mitogenic ER signaling throughout disease progression, including after acquired resistance to existing endocrine agents. Such continued dependence on ER signaling highlights the need for next generation therapies that more effectively block ER function in tumors. Fulvestrant was first discovered as a “pure antiestrogen”, in contrast to earlier generation ER therapeutic ligands that exhibit weak agonistic activity, such as tamoxifen. After its discovery as a full ER antagonist, fulvestrant was demonstrated to decrease ER protein levels through proteasome-mediated degradation. These observations led to the compelling hypothesis that elimination of ER by fulvestrant drives suppression of ER signaling. Importantly however, we recently demonstrated that fulvestrant and other full ER antagonists (e.g. GDC-0927) dramatically slow the intranuclear mobility of ER [Cell 178:4 (2019)]. We argue that such immobilization prevents ER function, and that increased ER turnover is a downstream consequence of immobilization, rather than a cause of ER inhibition. Here, we describe GDC-9545, our latest generation ER antagonist, currently being evaluated in clinic, that robustly immobilizes ER, and drives profound ER suppression in vivo. In the HCI-011 PDX model, we find that GDC-9545 can achieve greater ER pathway inhibition than can be achieved by tamoxifen. Intriguingly, although tamoxifen exhibits partial ER inhibition, likely through preventing recruitment of co-activators to the ER ligand binding domain, it drives increased accessibility at ~2500 chromatin sites, as determined by ATAC-seq. Chromatin regions exhibiting increased accessibility upon tamoxifen treatment are significantly enriched for the ERE motif, while a smaller number of sites exhibiting decreased accessibility upon treatment are enriched for AP-1 motifs. In contrast, GDC-9545 profoundly decreases chromatin accessibility at both ERE and AP-1 motifs, despite not fully eliminating ER protein. Notably, sites exhibiting decreased accessibility upon GDC-9545 treatment in the PDX in vivo, significantly overlap with sites displaying increased accessibility in estrogen-stimulated MCF7 cells in vitro. GDC-9545-treatment additionally alters accessibility at sites enriched for the FOXA1 motif, though unexpectedly, a sub-set of these sites exhibit increased accessibility while a distinct sub-set of sites exhibit decreased accessibility. We speculate that this particular pattern of accessibility changes may reflect redistribution of FOXA1 upon GDC-9545 treatment, and we will further explore this hypothesis. These data provide further insights into the impact of ER immobilization by the latest generation of pure antiestrogens. Citation Format: Ciara Metcalfe, Wei Zhou, Jane Guan, Robert A. Blake, Tom De Bruyn, Jennifer M. Giltnane, Ellen Ingalla, Tracy Kleinheinz, Jun Liang, Vidhi Mody, Jason Oeh, Savita Ubhayakar, Ingrid Wertz, Amy Young, Jason Zbieg, Xiaojing Wang, Marc Hafner. GDC-9545: A pure antiestrogen clinical candidate that immobilizes the estrogen receptor and profoundly alters chromatin accessibility in vivo [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3406.

Abstract DDT02-04: GDC-9545: Discovery of an oral estrogen receptor antagonist, degrader, and immobilizer for the treatment of ER+ breast cancer patients

Abstract GDC-9545: Discovery of an orally bioavailable pure antiestrogen for the treatment of ER+ breast cancer patients. Breast cancer affects one out of every eight women in their lifetime making it the most frequently occurring cancer among women. Mortality remains high with more than 40,000 of women expected to die of breast cancer in 2019 in the U.S alone. Approximately 70% of all breast cancer expresses estrogen receptor alpha (ERα), making it a prime target for the treatment of breast cancer. Despite effective endocrine therapies, many patients eventually relapse and become resistant to standard of care treatments. These endocrine resistant tumors often continue to depend on ERα for growth and survival, as evidenced by their sensitivity to the selective estrogen receptor antagonist and degrader, fulvestrant. However, fulvestrant is limited by its pharmaceutical and pharmacokinetic properties and its intramuscular route of administration to achieve sufficient target occupancy. Consequently, selective ERα ligands with desirable mechanisms of action and superior drug properties were sought after, allowing consistent and rapid achievement of therapeutic exposure. We have reported previously of our first and second generations of orally available ERα ligands, GDC-0810 and GDC-0927 respectively. Targeting a pure antiestrogen profile and guided by co-crystal structures, we identified a structurally distinct scaffold from these first two generations. Fine-tuning the physiochemical properties while balancing potency and efficacy culminated into a third generation development candidate GDC-9545. GDC-9545 immobilizes ERα to achieve full antagonism and degradation. It inhibits tumor growth or causes tumor regression in patient derived xenograft mouse models. It is orally bioavailable and has high safety margins in preclinical species. GDC-9545 is currently in active clinical testing. Citation Format: Xiaojing Wang, Xiaojing Wang. GDC-9545: Discovery of an oral estrogen receptor antagonist, degrader, and immobilizer for the treatment of ER+ breast cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr DDT02-04.

Discovery of Sulfated Small Molecule Inhibitors of Matrix Metalloproteinase-8.

Elevated matrix metalloproteinase-8 (MMP-8) activity contributes to the etiology of many diseases, including atherosclerosis, pulmonary fibrosis, and sepsis. Yet, very few small molecule inhibitors of MMP-8 have been identified. We reasoned that the synthetic non-sugar mimetics of glycosaminoglycans may inhibit MMP-8 because natural glycosaminoglycans are known to modulate the functions of various MMPs. The screening a library of 58 synthetic, sulfated mimetics consisting of a dozen scaffolds led to the identification of only two scaffolds, including sulfated benzofurans and sulfated quinazolinones, as promising inhibitors of MMP-8. Interestingly, the sulfated quinazolinones displayed full antagonism of MMP-8 and sulfated benzofuran appeared to show partial antagonism. Of the two, sulfated quinazolinones exhibited a >10-fold selectivity for MMP-8 over MMP-9, a closely related metalloproteinase. Molecular modeling suggested the plausible occupancy of the S1′ pocket on MMP-8 as the distinguishing feature of the interaction. Overall, this work provides the first proof that the sulfated mimetics of glycosaminoglycans could lead to potent, selective, and catalytic activity-tunable, small molecular inhibitors of MMP-8.

Onapristone Extended Release: Safety Evaluation from Phase I-II Studies with an Emphasis on Hepatotoxicity.

IntroductionAntiprogestins have demonstrated promising activity against breast and gynecological cancers, but liver-related safety concerns limited the advancement of this therapeutic class. Onapristone is a full progesterone receptor antagonist originally developed as an oral contraceptive and later evaluated in phase II studies for metastatic breast cancer. Because of liver enzyme elevations identified during clinical studies, further development was halted. Evaluation of antiprogestin pharmacology and pharmacokinetic data suggested that liver enzyme elevations might be related to off-target or metabolic effects associated with clinical drug exposure.ObjectiveWe explored whether the use of a pharmaceutic strategy targeting efficacious systemic dose concentrations, but with diminished peak serum concentrations and/or total drug exposure would mitigate hepatotoxicity. Twice-daily dosing of an extended-release formulation of onapristone was developed and clinically evaluated in light of renewed interest in antiprogestin therapy for treating progesterone receptor-positive breast and gynecologic cancers. The hepatotoxic potential of extended-release onapristone was assessed from two phase I–II studies involving patients with breast, ovarian, endometrial, and prostate cancer.ResultsAmong the 88 patients in two phase I–II studies in progesterone receptor-positive malignancies treated with extended-release onapristone, elevated alanine aminotransferase/aspartate aminotransferase levels were found in 20% of patients with liver metastases compared with 6.3% without metastases. Of five patients with grade 3 or higher alanine aminotransferase elevations with or without bilirubin elevations (four with breast cancer and one with endometrial cancer), four were assessed as unrelated to extended-release onapristone by the safety data review committee. Furthermore, while the fifth patient’s liver enzyme elevations were considered possibly drug related by the study investigator, they were adjudicated as unlikely to be related (< 25% likelihood) by a subsequent independent hepatologist.ConclusionsThese results suggest that the extended-release formulation by reducing drug exposure may be associated with a reduced risk of hepatotoxicity, and supports the continued clinical evaluation of extended-release onapristone for treating progesterone receptor-positive cancers.

Discovery of GNE-149 as a Full Antagonist and Efficient Degrader of Estrogen Receptor alpha for ER+ Breast Cancer.

Estrogen receptor alpha (ERα) is a well-validated drug target for ER-positive (ER+) breast cancer. Fulvestrant is FDA-approved to treat ER+ breast cancer and works through two mechanisms-as a full antagonist and selective estrogen receptor degrader (SERD)-but lacks oral bioavailability. Thus, we envisioned a "best-in-class" molecule with the same dual mechanisms as fulvestrant, but with significant oral exposure. Through lead optimization, we discovered a tool molecule 12 (GNE-149) with improved degradation and antiproliferative activity in both MCF7 and T47D cells. To illustrate the binding mode and key interactions of this scaffold with ERα, we obtained a cocrystal structure of 6 that showed ionic interaction of azetidine with Asp351 residue. Importantly, 12 showed favorable metabolic stability and good oral exposure. 12 exhibited antagonist effect in the uterus and demonstrated robust dose-dependent efficacy in xenograft models.

Preclinical profile and phase I clinical trial of a novel androgen receptor antagonist GT0918 in castration-resistant prostate cancer

PurposeWe conducted preclinical experiments and phase I clinical trial to investigate the safety, pharmacokinetics (PK) and antitumour effects of GT0918 in castration-resistant prostate cancer (CRPC). Experimental designAn androgen receptor (AR) competitive binding assay was performed, followed by evaluation of GT0918 on AR protein expression. The efficacy of GT0918 was investigated in a castration-resistant xenograft model. A phase I dose-escalation study of GT0918 in CRPC was also carried out to evaluate its safety, PK and antitumour efficacy. ResultsGT0918 was demonstrated to inhibit the binding of androgen to AR more potently than MDV3100, and to effectively reduce the AR protein level. GT0918 inhibited the transcriptional activity of wild-type AR and AR with clinically relevant ligand-binding domain mutations. Furthermore, GT0918 significantly inhibited the growth of prostate cancer. A total of 16 patients was treated with GT0918 at five dose levels. Among these 16 patients, 10 and 2 patients, respectively, completed a three-cycle and six-cycle treatment, in which MTD was not reached. All the treatment-related adverse events were grade I, including hypercholesterolemia, hypertriglyceridemia, fatigue and anaemia. PK parameters showed that drug exposure increased with dose proportionally from 50 to 300 mg and a saturation was observed between 300 and 400 mg. PSA declines of ≥30% and ≥50% were, respectively, observed in six and two cases. All the 12 patients with metastatic soft tissue lesions confirmed stable disease. ConclusionsGT0918, a full AR antagonist without agonist effect, has high binding affinity to AR with AR protein down-regulation activity. GT0918 is demonstrated to be well tolerated with a favourable PK profile and exhibits promising antitumour activity in CRPC. Clinicaltrialsgov identifier CTR20150501.

Basket study of the oral progesterone antagonist onapristone ER in women with progesterone receptor positive (PR+) recurrent granulosa cell tumor (GCT), low-grade serous ovarian cancer (LGSOC), or endometrioid endometrial cancer (EEC).

TPS6098 Background: Onapristone extended release (ER) is a type I full progesterone antagonist that inhibits progesterone mediated PR activation and stabilizes PR association with corepressors. Onapristone has shown activity across multiple preclinical models of hormonally driven cancer. A phase I dose escalation study of onapristone ER in PR+ breast, endometrial and ovarian cancer patients found all doses tested to be well tolerated, with 50mg PO BID determined to be the recommended phase 2 dose (RP2D). GCT (98% of cases PR+), LGSOC (58% of cases PR+) and EEC (67% of cases PR+) are hormonally driven cancers which generally have poor responses to chemotherapy and limited treatment options in the recurrent setting. Methods: This is an open-label, investigator-initiated basket study of onapristone ER in patients with PR+ recurrent GCT, LGSOC, or EEC currently enrolling patients at Memorial Sloan Kettering Cancer Center in NY, USA (NCT03909152). The primary objective is to evaluate the efficacy, in terms of response rate by RECIST 1.1 criteria, within 36 weeks of treatment. Eligible patients must have received at least 1 prior line of chemotherapy, have measurable disease by RECIST 1.1 criteria, and have tumor tissue collected within 3 years prior to enrollment with PR expression ≥ 1% by IHC. Patients are allowed to have unlimited additional prior lines of chemotherapy, biologic therapy, immunotherapy or hormonal therapy. Enrolled patients are treated with onapristone ER 50mg PO BID until time of progression or intolerable toxicity. The 3 disease cohorts are currently enrolling to Stage I in parallel with expansion from stage I to stage II planned when the prespecified response criteria are met for each cohort as described in the table below. Clinical trial information: NCT03909152. [Table: see text]

OR12-07 Full Antagonism of Breast Cancer Cell Proliferation Can Result from Many Ligand-Induced Conformational Distortions of the Estrogen Receptor Ligand Binding Domain

Although most estrogen receptor alpha (ERα)-positive breast cancers initially respond well to endocrine therapies using aromatase inhibitors (AIs) or antiestrogens, after varying time periods the cancer frequently recurs as metastatic disease. A significant fraction of these recurrences are driven by ERs that have acquired activating mutations in their ligand binding domains (LBDs), giving them constitutive activity and thus resistance to AIs. Because these mutations also reduce the affinity and potency of SERMs and SERDs, expanded efforts have been made to vary the structure of antiestrogens to make them more potent.Typical antiestrogens are comprised of a core element that binds securely in the ligand binding pocket and from which extends a single ring (ring E) having a side chain that sterically interferes with the position of helix-12 by direct antagonism, reorienting it so that it occludes the activation function 2 (AF2) hydrophobic groove for coactivator binding. Through structural studies, we found that bridged oxabicycloheptene-sulfonamide (OBHS-N) core ligands have two rings (E and F) that can be poised to engage in both “direct antagonism” and “indirect antagonism”, the latter of which disrupts the orientation of helix-12 by impinging on helix-11 and the helix-11–12 loop.In this study, we have placed typical antiestrogen side chains on either the E or the F ring of OBHS-N core ligands and characterized their activities in ERα-positive breast cancer cells. All compounds have full antiproliferative activity and reverse estrogen-regulated gene expression, with the antiproliferative potency of each type of side chain having a distinct preference for E- vs F-ring attachment. Conformational analysis using a multiplexed coregulator peptide interaction assay shows that compounds with an E-ring substitution have interaction profiles similar to 4-hydroxytamoxifen and fulvestrant, whereas the F-ring substitution gives a very different pattern, suggesting that the antagonist activity of the two classes rely on different sets of coregulator proteins. A large number of high resolution (better than 2 Å) X-ray crystal structures reveal that this set of novel ER antagonists disrupt the conformation of the ER LBD in a variety of ways, several of which are distinct from those seen with previous antiestrogens such as Tamoxifen and Fulvestrant.Our findings expand design concepts by which ERα ligands can block the activity of this receptor and illustrate how direct and indirect modes of ER antagonism can be combined to facilitate the development of more efficacious antiestrogens for breast cancer treatment and possibly for regulating ER-mediated activities in other estrogen target tissues.

Syringaresinol as a novel androgen receptor antagonist against wild and mutant androgen receptors for the treatment of castration-resistant prostate cancer: molecular docking, in-vitro and molecular dynamics study

Phytoestrogens are dietary estrogens having similar structure as of estrogen. Some of these phytoestrogens are androgen receptor (AR) antagonists and exhibit preventive role in the prostate cancer. However, in androgen-independent prostate cancer (AIPC) the ARs were mutated (T877A, W741L, F876L, etc.) and these mutant ARs convert the antagonist to agonist. Our aim in this study is to find phytoestrogens that could function as an antagonist with wild and mutant ARs. The phytoestrogens were analyzed for binding affinity with wild and mutant ARs in agonist and antagonist conformations. The point mutations were carried out using Chimera. The antagonist AR conformation was modeled using Modeller. We hypothesize that the compounds having binding affinity with agonist AR conformation could not function as a full or pure antagonist. Most of the phytoestrogens have binding affinity with agonist AR conformation contradicting previous results. For example, genistein which is a widely studied isoflavone has known AR antagonist property. However, in our study, it had good binding affinity with agonist AR conformation. Hence, to confirm our hypothesis, we tested genistein in LNCaP (T877A mutant AR) cells by qPCR studies. The genistein functioned as an antagonist only in the presence of an androgen indicting a partial agonist type of activity. The in-vitro results supported our docking hypothesis. We applied this principle and found syringaresinol could function as an antagonist with wild and mutated ARs. Further, we carried out molecular dynamics for the hit molecule to confirm its antagonist binding mode with mutant AR.Communicated by Ramaswamy H. Sarma

P6346Purinergic receptor Y6 (P2Y6) deficiency impairs left ventricular function

Abstract Introduction Cardiomyopathies due to pathological remodelling are the most common myocardial disorders and leading indication for heart transplant in young adults. Cardiomyocytes rely on sessile macrophage phagocytosis for cell homeostasis. Purinergic receptor Y6, selectively binding to UDP, promotes phagocytosis and is involved in several tissue-remodelling processes. We hypothesize a critical role of P2Y6 in the maintenance of cardiac tissue homeostasis in vivo. Methods Echocardiography was performed on male C57Bl6 wild-type (WT) and P2Y6-deficient (KO) mice at different ages up to 20 weeks. In addition, WT and KO mice underwent bone marrow transplantation. For histological analysis, hearts from WT and KO mice were stained with HE, Masson's trichrome, wheat germ agglutinin, CD11b, LC3B and TUNEL. Cardiac ultrastructure of WT and KO hearts was investigated by electron microscopic imaging. Furthermore, uptake of fluorescent pHrodo bioparticles by WT and KO bone marrow derived macrophages (BMDMs), either in presence or absence of specific P2Y6 agonist UDP or full antagonist MRS2578, was measured in vitro. Results KO mice were significantly impaired in their LV function by reduced ejection fraction (WT: 57.37±1.27%, n=13, KO: 44.13±1.09%, n=16, p&lt;0.0001), stroke volume (WT+: 39.25±1.94μl, n=13, KO: 33.57±1.94μL, n=16, p&lt;0.05), and cardiac output (WT: 20.79±1.11 mL/min, n=13, KO: 17.84±0.85 mL/min, n=16, p&lt;0.05). A long-term follow-up revealed progressive impairment of the cardiac function (4 w: WT: 62.07±1.11%, n=5, KO: 48.73±1.03%, n=10, p&lt;0.0001; 6 w: WT: 54.29±1.88%, n=5, KO: 44.61±1.4%, n=10, p&lt;0.01; 8 w: WT: 56.43±1.44%, n=5, KO: 44.72±0.89%, n=10, p&lt;0.0001). Echocardiography 6 weeks after bone marrow transplantation demonstrated convalescence upon reconstitution with WT bone marrow (WT in KO: 46.19±2.68%, n=3 vs KO in WT: 38.40±1.26%, n=3). No major differences with regard to morphology, cell size, collagen deposition, leukocyte infiltration or apoptosis were observed in histology. However, LC3B expression was increased in KO mice (mean fluorescent area: WT: 191.1±19.93μm2, n=5, KO: 261.2±18.34μm2, n=10, p&lt;0.05). Electron microscopic imaging revealed a distinctly impaired ultrastructure with T-tubule remodelling, mitochondrial derangement and abnormal numbers of autophagosomes in KO hearts. In vitro, fluorescent particle phagocytosis by BMDMs was completely blocked after treatment with MRS2578. Conclusion Global P2Y6 deficiency leads to a progressive cardiomyopathy in mice, mainly characterized by an impaired left ventricular function and ultrastructural irregularities. Its exacerbation seems to be prevented by reconstitution with WT bone marrow. For the underlying mechanism, we conclude a deranged cardiomyocyte homeostasis in KO animals due to defective phagocytic activity of resident macrophages. Potential induction of cardiac clearance via P2Y6 signalling could be a promising therapeutic target in the field of cardiomyopathies.

Differential activity of BPA, BPAF and BPC on zebrafish estrogen receptors in vitro and in vivo

The high volume production compound bisphenol A (BPA) is of environmental concern largely because of its estrogenic activity. Consequently, BPA analogues have been synthesized to be considered as replacement molecules for BPA. These analogues need to be thoroughly evaluated for their estrogenic activity. Here, we combined mechanism zebrafish-based assays to examine estrogenic and anti-estrogenic activities of BPA and two of its analogues, bisphenol AF (BPAF) and bisphenol C (BPC) in vitro and in vivo. In vitro reporter cell lines were used to investigate agonistic and antagonistic effects of the three bisphenols on the three zebrafish estrogen receptors. The transgenic Tg(5 × ERE:GFP) and Cyp19a1b-GFP zebrafish lines were then used to analyze estrogenic and anti-estrogenic responses of the three bisphenols in vivo. BPA, BPAF and BPC were agonists with different potencies for the three zebrafish estrogen receptors in vitro. The potent zfERα-mediated activity of BPA and BPAF in vitro resulted in vivo by activation of GFP expression in zebrafish larvae in the heart (zfERα-dependent) at lower concentrations, and in the liver (zfERβ-dependent) at higher concentrations. BPC induced zfERβ-mediated luciferase expression in vitro, and the zfERβ agonism led to activation of GFP expression in the liver and the brain in vivo. In addition, BPC acted as a full antagonist on zfERα, and completely inhibited estrogen-induced GFP expression in the heart of the zebrafish larvae. To summarize, applying a combination of zebrafish-based in vitro and in vivo methods to evaluate bisphenol analogues for estrogenic activity will facilitate the prioritization of these chemicals for further analysis in higher vertebrates as well as the risk assessment in humans.

The Cattle Fever Tick, Rhipicephalus microplus, as a Model for Forward Pharmacology to Elucidate Kinin GPCR Function in the Acari.

The success of the acaricide amitraz, a ligand of the tick tyramine/octopamine receptor (a G protein-coupled receptor; GPCR), stimulated interest on arthropod-specific GPCRs as targets to control tick populations. This search advances tick physiology because little is known about the pharmacology of tick GPCRs, their endogenous ligands or their physiological functions. Here we explored the tick kinin receptor, a neuropeptide GPCR, and its ligands. Kinins are pleiotropic insect neuropeptides but their function in ticks is unknown. The endogenous tick kinins are unknown and their cDNAs have not been cloned in any species. In contrast, more than 271 insect kinin sequences are available in the DINeR database. To fill this gap, we cloned the kinin cDNA from the cattle fever tick, Rhipicephalus microplus, which encodes 17 predicted kinins, and verified the kinin gene structure. We predicted the kinin precursor sequences from additional seven tick species, including Ixodes scapularis. All species showed an expansion of kinin paracopies. The “kinin core” (minimal active sequence) of tick kinins FX1X2WGamide is similar to those in insects. Pro was predominant at the X2 position in tick kinins. Toward accelerating the discovery of kinin function in ticks we searched for novel synthetic receptor ligands. We developed a dual-addition assay for functional screens of small molecules and/or peptidomimetics that uses a fluorescent calcium reporter. A commercial library of fourteen small molecules antagonists of mammalian neurokinin (NK) receptors was screened using this endpoint assay. One acted as full antagonist (TKSM02) with inhibitory concentration fifty (IC50) of ∼45 μM, and three were partial antagonists. A subsequent calcium bioluminescence assay tested these four antagonists through kinetic curves and confirmed TKSM02 as full antagonist and one as partial antagonist (TKSM14). Antagonists of NK receptors displayed selectivity (>10,000-fold) on the tick kinin receptor. Three peptidomimetic ligands of the mammalian NK receptors (hemokinin 1, antagonist G, and spantide I) were tested in the bioluminescence assay but none were active. Forward approaches may accelerate discovery of kinin ligands, either as reagents for tick physiological research or as lead molecules for acaricide development, and they demonstrate that selectivity is achievable between mammalian and tick neuropeptide systems.

CD40/anti-CD40 antibody complexes which illustrate agonist and antagonist structural switches

BackgroundCD40 is a 48 kDa type I transmembrane protein that is constitutively expressed on hematopoietic cells such as dendritic cells, macrophages, and B cells. Engagement of CD40 by CD40L expressed on T cells results in the production of proinflammatory cytokines, induces T helper cell function, and promotes macrophage activation. The involvement of CD40 in chronic immune activation has resulted in CD40 being proposed as a therapeutic target for a range of chronic inflammatory diseases. CD40 antagonists are currently being explored for the treatment of autoimmune diseases and several anti-CD40 agonist mAbs have entered clinical development for oncological indications.ResultsTo better understand the mode of action of anti-CD40 mAbs, we have determined the x-ray crystal structures of the ABBV-323 (anti-CD40 antagonist, ravagalimab) Fab alone, ABBV-323 Fab complexed to human CD40 and FAB516 (anti-CD40 agonist) complexed to human CD40. These three crystals structures 1) identify the conformational CD40 epitope for ABBV-323 recognition 2) illustrate conformational changes which occur in the CDRs of ABBV-323 Fab upon CD40 binding and 3) develop a structural hypothesis for an agonist/antagonist switch in the LCDR1 of this proprietary class of CD40 antibodies.ConclusionsThe structure of ABBV-323 Fab demonstrates a unique method for antagonism by stabilizing the proposed functional antiparallel dimer for CD40 receptor via novel contacts to LCDR1, namely residue position R32 which is further supported by a closely related agonist antibody FAB516 which shows only monomeric recognition and no contacts with LCDR1 due to a mutation to L32 on LCDR1. These data provide a structural basis for the full antagonist activity of ABBV-323.

A new series of bicalutamide, enzalutamide and enobosarm derivatives carrying pentafluorosulfanyl (SF5) and pentafluoroethyl (C2F5) substituents: Improved antiproliferative agents against prostate cancer

SAR studies on bicalutamide, enobosarm and enzalutamide analogues, functionalised with polyfluorinated groups, is presented. Among the novel bicalutamide and enobosarm derivatives synthesised, several displayed significantly improved in vitro anticancer activity, with IC50 values in the low micromolar range against four different prostate cancer cell lines (LNCaP, VCaP, DU-145 and 22Rv1), showing up to 48-fold increase in comparison with the parent structures. In particular, SF5 enobosarm analogues were found to be most potent compounds, full AR antagonists and with favourable ADME properties. The most promising compound (48a) was evaluated for its in vivo efficacy in PC xenograft mouse model (22Rv1) with results comparable to the standard-of-care docetaxel.

Abstract 1004: Leveraging an expanding tool box for ER+ breast cancer: exploring breast cancer dependencies to support optimal therapeutic strategies

Abstract Aromatase inhibitors (AIs), the Estrogen Receptor (ER) modulator tamoxifen and the full ER antagonist fulvestrant each target ER signaling and constitute the backbone of standard of care for ER+ breast cancer. The growing appreciation of liabilities associated with each of these modalities, which may limit their clinical potential, has triggered a wave of activity around next-generation ER therapeutics, and there are currently more than 10 new molecular entities targeting ER being evaluated in Phase 1 clinical trials. In addition to a collection of ER-targeted therapeutics either approved or under evaluation, the emergence of CDK4/6 inhibitors as combination partners has meaningfully contributed to the oncologist’s tool box for the treatment of breast cancer, while also adding complexity to the treatment landscape. Although direct ER antagonists are expected to be superior to AIs in patients with tumors expressing constitutively activating ER mutations (e.g. ER.Y537S), it is currently unclear if a subset of ER wildtype patients may likewise derive superior benefit from direct ER targeting. It is also unclear which ER+ tumors require CDK4/6 inhibitor/ER antagonist combinations for maximal anti-tumor activity, versus those where single-agent ER antagonist treatment may achieve full cell cycle control. To address these key questions, we used a panel of 22 ER+ cell lines to evaluate the relative anti-proliferative and cytotoxic effects of estrogen (E2) withdrawal (modeling AIs), ER modulation, and full ER inhibition, as single agents and in combination with the CDK4/6 inhibitor Palbociclib. We find a number of cell lines in which direct ER antagonism is superior to E2 withdrawal and is associated with E2-independent ER signaling not attributable to ER mutations. The impact of E2 withdrawal on cell cycle control and proliferation is significantly enhanced by concurrent CDK4/6 inhibition, in line with clinical observations, however, direct ER antagonism in combination with CDK4/6 inhibition remains most efficacious. We are integrating our cell response data with detailed molecular analyses and transcriptional profiling to 1) identify cellular contexts, beyond ER mutations, where direct ER antagonism is superior to targeting E2 synthesis, 2) identify tumors where full cell cycle control and anti-tumor efficacy is achievable through single-agent ER targeting, and 3) better understand the basis for the co-operativity of endocrine suppression with CDK4/6 inhibition. Deconvolution of the ER+ breast cancer response to distinct modes of ER signaling inhibition alone and in combination with CDK4/6 inhibitors has the potential to guide clinical decision-making for personalized patient care. Citation Format: Wei Zhou, Jane Guan, Georgia Hatzivassiliou, Anneleen Daemen, Marc Hafner, Ciara Metcalfe. Leveraging an expanding tool box for ER+ breast cancer: exploring breast cancer dependencies to support optimal therapeutic strategies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1004.

Abstract 218: AVID100 is an anti-EGFR ADC that promotes DM1-meditated cytotoxicity on cancer cells but not on normal cells

Abstract AVID100 is a novel rationally designed antibody drug conjugate (ADC) that specifically targets the epidermal growth factor receptor (EGFR). EGFR is highly expressed on a variety of cancers making it a promising target for ADCs. However, due to the presence of EGFR on normal skin cells, on-target off-tumor toxicity is a concern. AVID100 is an anti-EGFR-DM1 conjugate exhibiting high potency against cancer cells relative to unconjugated antibodies, while not exhibiting increased toxicity on normal cells. In a series of in vitro and in vivo experiments, we provide mechanistic evidence rationalizing why AVID100 shows higher cytotoxicity on tumor cells compared to normal cells. The antibody moiety of AVID100, denoted MAB100, was shown to have a high affinity for EGFR (approximately 2nM) and to compete with EGF for binding to the EGFR. Also, MAB100 prevented downstream signalling from EGFR. In addition to exhibiting full antagonist activity, MAB100 effectively delivered the microtubule inhibitor DM1 to tumor cells, as demonstrated by an increase in apoptosis in AVID100-treated tumor cells relative to MAB100-treated tumor cells. AVID100 was very effective on tumor cell lines derived from breast, head and neck, and lung cancers with the cytotoxic IC50 values generally correlating with the number of EGFR molecules on the cell surface. Importantly, on keratinocytes, AVID100 did not exhibit increased cytotoxicity relative to MAB100. We hypothesized that this lack of increase in cytotoxicity on keratinocytes is due to the antagonistic effect of MAB100 on EGFR signalling. Blocking the EGFR pathway in keratinocytes strongly inhibits their proliferation, which should protect them against the cytotoxic action of DM1. To address this hypothesis, we compared the effect of AVID100 to that of a non-antagonistic anti-EGFR ADC, huML66-DM1. As expected, both ADCs were highly effective at killing MDA-MB-468 cancer cells, confirming their ability to deliver DM1. In contrast, keratinocytes exhibited a significantly higher level of apoptosis in response to huML66-DM1 as compared to AVID100. These results confirm that the antagonistic nature of the antibody moiety of AVID100 plays a critical role in protecting keratinocytes from the cytotoxic effect of DM1. Finally, the activity of AVID100 was investigated in multiple mouse xenograft studies, including in MDA-MB-468 human breast adenocarcinoma, H292 NSCLC, and FaDu SSCHN models. AVID100 treatment significantly reduced tumor growth and caused tumor regressions in some of the mice, even when administered as a single dose. Toxicology studies in cynomolgus monkeys demonstrated that AVID100 was well tolerated at up to 4 weekly doses of 10mg/kg. AVID100 has completed a Phase 1 clinical trial with the recommended Phase 2 dose having been determined. The Phase 2 trial of AVID100 is ongoing. Citation Format: Michael J. Thwaites, Rene Figueredo, Gilles Tremblay, James Koropatnick, Victor Goldmacher, Maureen O’Connor-McCourt. AVID100 is an anti-EGFR ADC that promotes DM1-meditated cytotoxicity on cancer cells but not on normal cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 218.

Abstract 2625: Identification of CTX-30916 as a novel antagonist of progesterone receptor signaling pathways

Abstract Breast cancer is the most commonly diagnosed cancer in women and the second leading cause of cancer related death in women. Progesterone is a major mitogen in the adult human mammary epithelium that, in addition to estrogen, is a key driver of breast cancer cell proliferation. Activated PR signaling through MAPK-driven phosphorylation of the progesterone receptor (PR) is also an important driver of breast cancer stem cells. PR expression is associated with the presence of estrogen receptor-alpha (ESR1) mutations that are constitutively active and linked to resistance in the clinic in response to aromatase inhibitors, antiestrogens and CDK4/6 inhibitors. Thus, PR may play a role in tumor relapse and resistance in the clinic and blocking both estrogen and progesterone receptors would likely be an effective approach in 2L treatment for patients with advanced breast cancer. There is great interest in evaluating antiprogestins in the clinic in combination with antiestrogens or CDK4/6 inhibitors. Apristor®, an extended release formulation of onapristone, a full antagonist of PR, that has efficacy in multiple in vivo and in vitro models in combination with antiestrogestins and CDK4/6 inhibitors, is currently being evaluated in several human clinical trials. In this study we have examined the in vitro properties of CTX-30916, a novel mono-N-desmethyl analog of onapristone. CTX-30916 binds potently to PR and acts as an antagonist in cell-based transfection assays. CTX-30916 displays selectivity against other steroid receptors in binding assays and is also a potent inhibitor of T47D breast cancer cell proliferation. Interestingly, CTX-30916 leads to a unique co-regulator binding pattern in the Microarray Assay for Real-time Co-regulator-Nuclear receptor interaction (MARCoNI) assay compared with onapristone and mifepristone indicating that the co-regulator peptide-PR interaction landscape is different between the various antiprogestins. These data suggest that CTX-30916 binding to PR leads to a distinct receptor conformation and antagonizes PR signaling pathways in cancer through a unique mechanism of action compared to other antiprogestins. Citation Format: Deepak Lala, Tasir Haque. Identification of CTX-30916 as a novel antagonist of progesterone receptor signaling pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2625.

Predicting estrogen receptor binding of chemicals using a suite of in silico methods – Complementary approaches of (Q)SAR, molecular docking and molecular dynamics

With the aim of obtaining reliable estimates of Estrogen Receptor (ER) binding for diverse classes of compounds, a weight of evidence approach using estimates from a suite of in silico models was assessed. The predictivity of a simple Majority Consensus of (Q)SAR models was assessed using a test set of compounds with experimental Relative Binding Affinity (RBA) data. Molecular docking was also carried out and the binding energies of these compounds to the ERα receptor were determined. For a few selected compounds, including a known full agonist and antagonist, the intrinsic activity was determined using low-mode molecular dynamics methods. Individual (Q)SAR model predictivity varied, as expected, with some models showing high sensitivity, others higher specificity. However, the Majority Consensus (Q)SAR prediction showed a high accuracy and reasonably balanced sensitivity and specificity. Molecular docking provided quantitative information on strength of binding to the ERα receptor. For the 50 highest binding affinity compounds with positive RBA experimental values, just 5 of them were predicted to be non-binders by the Majority QSAR Consensus. Furthermore, agonist-specific assay experimental values for these 5 compounds were negative, which indicates that they may be ER antagonists. We also showed different scenarios of combining (Q)SAR results with Molecular docking classification of ER binding based on cut-off values of binding energies, providing a rational combined strategy to maximize terms of toxicological interest.

Selective stimulation of central GABAAα2,3,5 receptors increases intake and motivation to consume sucrose solution in rats

Benzodiazepines are one of the most commonly prescribed anxiolytic drugs in America, and between 2006 and 2015 prescription rates increased by an estimated 27.1%. Weight gain is a common side effect of these drugs and it may result from increased feeding caused by drug-enhanced food palatability. We investigated the role of specific GABAA receptor subtypes involved with benzodiazepine-induced food consumption through third ventricle injections of L-838,417, a partial agonist of GABAA α2, α3, and α5 subunits, and a full antagonist of the α1 receptor subunit. A microanalysis of the licking behavior of adult male rats to a sucrose solution was used to isolate drug effects on specific consummatory behaviors that include: hedonic taste evaluation, food approach behavior, and oromotor function. L-838,417 dose-dependently increased intake through increases in the motivation to approach the solution (shorter pause intervals between bouts of licking) and through enhancement of measures associated with hedonic taste evaluation. Oromotor depressant effects previously associated with broad-spectrum benzodiazepine receptor agonists were not observed. These results indicate that nuclei in proximity to the ventricles respond to GABAA α2, α3, or α5 activation to induce motivation to feed, absent of α1 receptor subunit activation. Furthermore, activation of the α1 subunit is not necessary for benzodiazepine hyperphagia and may instead contribute to the oromotor depressant and sedative properties of classic benzodiazepine agonists. Hypothalamic nuclei such as the paraventricular nucleus may be involved in the benzodiazepine-increased motivation to feed, while the parabrachial nucleus of the hindbrain could contribute to benzodiazepine-induced enhancement of taste palatability.

Novel Dopamine D4 Receptor‐Selective Compounds Reveal Structure‐Activity Relationships that Engender Agonist Efficacy

The dopamine D4 receptor (D4R) plays important roles in cognition, attention, and decision making. Novel D4R‐selective ligands have promise in medication development for neuropsychiatric conditions, including Alzheimer's disease and substance use disorders. To identify new D4R‐selective ligands, and to understand the molecular determinants of agonist efficacy at D4R, we report a series of eighteen novel ligands based on the classical D4R agonist A‐412997 (2‐(4‐(pyridin‐2‐yl)piperidin‐1‐yl)‐N‐(m‐tolyl)acetamide). We report a series of eighteen novel D4R ligands with varied substitutions on the phenylpiperidinyl ring, linker region, and/or the arylamide moieties. Compounds were profiled using radioligand binding displacement assays, β‐arrestin recruitment assays, cAMP inhibition assays, and molecular dynamics computational modeling. We identified several novel high‐affinity (Ki ≤ 4.3 nM) and D4R‐selective (&gt;100‐fold vs other D2‐like receptors) compounds with diverse partial agonist and antagonist profiles, falling into three structural groups. Group 1 modifications resulted in a gain of efficacy at D2Rs and D3Rs. Group 2 modifications resulted in lower partial agonist efficacy at D4R. Group 3 modifications resulted in high‐potency full antagonists at D4R. These compounds inform receptor‐ligand interactions that control efficacy at D2‐like receptors and may provide insights to targeted drug discovery leading to new compound development and new tools for a better understanding of the role of D4Rs in neuropsychiatric disorders.Support or Funding InformationSupport for this research was provided by High Point University, Fred Wilson School of Pharmacy. Additional support was provided by the National Institute of Neurological Disorders and Stroke‐Intramural Research Program, National Institute on Drug Abuse‐Intramural Research Program (Z1A DA000609), and Rowan University.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.