Tae-Kyung Lee

Peptidomimetic targeting of critical androgen receptor-coregulator interactions in prostate cancer.

The growth of advanced prostate cancer depends on androgen receptor signalling, however treatment options are limited. Here we report the disruption of specific protein-protein interactions involving LXXLL motifs in androgen receptor-coregulator proteins such as PELP1 using a novel, small molecule peptidomimetic (D2). D2 is stable, non-toxic and efficiently taken up by prostate cancer cells. Importantly, D2 blocks androgen-induced nuclear uptake and genomic activity of the androgen receptor. Furthermore, D2 abrogates androgen-induced proliferation of prostate cancer cells in vitro with an IC50 of 40 nM, and inhibits tumour growth in a mouse xenograft model. D2 also disrupts androgen receptor-coregulator interactions in ex vivo cultures of primary human prostate tumours. These findings provide evidence that targeting androgen receptor-coregulator interactions using peptidomimetics may be a viable therapeutic approach for patients with advanced prostate cancer.

Solid-phase synthesis of tris-benzamides as α-helix mimetics

Small molecules mimicking α-helices are of great interest since numerous protein-protein interactions use helical structures at the interface. With a goal of generating libraries of α-helix mimetics, an efficient solid-phase synthetic method was developed to produce tris-benzamides. The tris-benzamide scaffold was designed to place three side-chain functional groups found at the i, i + 4, and i + 7 positions of an α-helix, emulating one helical face. The synthetic strategy involves simple and iterative reactions of removal of an allyl ester, formation of an amide bond via an O → N acyl migration, and an O-alkylation. A small library of twenty tris-benzamides containing a variety of functional groups was prepared in high purity (83-99%) to demonstrate the versatility of the synthetic approach. This methodology allowed the facile and rapid construction of α-helix mimetics that would facilitate the identification of small molecules for target proteins.

Mouse Spermatogenesis Requires Classical and Nonclassical Testosterone Signaling

ABSTRACT Testosterone acts though the androgen receptor in Sertoli cells to support germ cell development (spermatogenesis) and male fertility, but the molecular and cellular mechanisms by which testosterone acts are not well understood. Previously, we found that in addition to acting through androgen receptor to directly regulate gene expression (classical testosterone signaling pathway), testosterone acts through a nonclassical pathway via the androgen receptor to rapidly activate kinases that are known to regulate spermatogenesis. In this study, we provide the first evidence that nonclassical testosterone signaling occurs in vivo as the MAP kinase cascade is rapidly activated in Sertoli cells within the testis by increasing testosterone levels in the rat. We find that either classical or nonclassical signaling regulates testosterone-mediated Rhox5 gene expression in Sertoli cells within testis explants. The selective activation of classical or nonclassical signaling pathways in Sertoli cells within testis explants also resulted in the differential activation of the Zbtb16 and c-Kit genes in adjacent spermatogonia germ cells. Delivery of an inhibitor of either pathway to Sertoli cells of mouse testes disrupted the blood-testis barrier that is essential for spermatogenesis. Furthermore, an inhibitor of nonclassical testosterone signaling blocked meiosis in pubertal mice and caused the loss of meiotic and postmeiotic germ cells in adult mouse testes. An inhibitor of the classical pathway caused the premature release of immature germ cells. Collectively, these observations indicate that classical and nonclassical testosterone signaling regulate overlapping and distinct functions that are required for the maintenance of spermatogenesis and male fertility.

Controllable core-shell-type resin for solid-phase peptide synthesis.

A simple, mild, and inexpensive biphasic functionalization approach is attempted for preparing an ideal core-shell-type resin. The core-shell-type architecture was constructed by coupling Fmoc-OSu to the amino groups on the shell layer of an aminomethyl polystyrene (AM PS) resin. The shell layer thickness of the resin could be easily controlled under mild conditions, which was characterized by confocal laser scanning microscopy (CLSM). The efficiency of core-shell-type resin for solid-phase peptide synthesis (SPPS) was demonstrated by the synthesis of various peptides and compared with commercially available noncore-shell-type resins such as AM PS and poly(ethylene glycol)-based resins. The core-shell-type resin provided effective performance during the synthesis of hydrophobic peptide sequences, a disulfide-bridged cyclic peptide, and a difficult PNA sequence. Furthermore, a highly aggregative peptide fragment, MoPrP 105-125, was synthesized more efficiently on the core-shell-type resin under microwave conditions than AM PS and ChemMatrix resins.

Estrogen receptor coregulator binding modulators (ERXs) effectively target estrogen receptor positive human breast cancers

The majority of human breast cancer is estrogen receptor alpha (ER) positive. While anti-estrogens/aromatase inhibitors are initially effective, resistance to these drugs commonly develops. Therapy-resistant tumors often retain ER signaling, via interaction with critical oncogenic coregulator proteins. To address these mechanisms of resistance, we have developed a novel ER coregulator binding modulator, ERX-11. ERX-11 interacts directly with ER and blocks the interaction between a subset of coregulators with both native and mutant forms of ER. ERX-11 effectively blocks ER-mediated oncogenic signaling and has potent anti-proliferative activity against therapy-sensitive and therapy-resistant human breast cancer cells. ERX-11 is orally bioavailable, with no overt signs of toxicity and potent activity in both murine xenograft and patient-derived breast tumor explant models. This first-in-class agent, with its novel mechanism of action of disrupting critical protein-protein interactions, overcomes the limitations of current therapies and may be clinically translatable for patients with therapy-sensitive and therapy-resistant breast cancers. DOI: http://dx.doi.org/10.7554/eLife.26857.001

Abstract 4148: Novel ERX-11 and CDK4/6 inhibitor combination therapy for treating therapy resistant breast cancer

BACKGROUND: The majority of the breast cancer is estrogen receptor alpha (ESR1) positive. While tamoxifen and letrozole therapies are effective, therapy resistance is common. Importantly, both therapy-sensitive and therapy-resistant tumors retain ESR1 signaling, via interaction with critical oncogenic coregulator proteins. Further, resistant tumors commonly acquire cyclin D1:CDK4/6 signaling via multiple mechanisms, cyclin D1 can independently activate ESR1 and thus contribute to estrogen independence of ESR+ tumor. Currently, CDK4/6 inhibitors in clinical trials for treating breast cancer, however, considering complex signaling interplay of estrogen and CDK axis, combination therapy of CDK inhibitor with other potent ESR1 targeted agents may have better utility and may prevent development of resistance to the CDK4/6 inhibitors. We recently developed a small organic molecule, ESR1 coregulator binding inhibitor ERX-11 (EtiraRx-11). The objective of this study is to test the utility of novel combination therapy of ERX-11 with CDK4/6 inhibitor palbociclib in treating therapy resistant cancer. METHODS: We have utilized multiple therapy sensitive and therapy-resistant models with various genetic back grounds. We tested efficacy using both acquired resistance and engineered models that express ESR1 mutations or oncogenes. Efficacy of combination therapy was tested using established in vitro assays including, MTT, colony formation, apoptosis, and cell cycle progression. Mechanistic studies were conducted using reporter gene assays, gene expression and signaling alterations. Xenograft studies were used to determine the in vivo efficacy of the combination therapy. RESULTS: ERX-11 effectively blocked ESR1-mediated oncogenic signaling and has potent anti-proliferative activity against therapy-sensitive and therapy-resistant breast cancer cells. Mechansistic studies showed that ERX-11 blocks the interaction between a subset of coregulators with both native and mutant forms of ESR1. ERX-11 showed potent activity in both preclinical xenograft models and patient-derived breast tumor explant models. Co-treatment of ERX-11 with palbociclib synergistically reduced cell viability and induced apoptosis of therapy sensitive and resistant breast cancer model cells. Importantly, combination therapy of ERX-11 and the palbociclib synergistically reduced the growth and induced apoptosis of tamoxifen and letrozole resistant xenograft tumors compared to either drug alone. Mechanistic studies showed combination therapy significantly altered E2F1 and ESR1 signaling pathways and promoted apoptosis. CONCLUSIONS: Collectively our studies have discovered a novel combinational treatment with ERX-11 and palbociclib for patients with therapy-sensitive and therapy-resistant breast cancers. Citation Format: Suryavathi Viswanadhapalli, Gangadhara Reddy Sareddy, Shi-Hong Ma, Tae-Kyung Lee, Rajeshwar Rao Tekmal, Jung-Mo Ahn, Ganesh Raj, Ratna K. Vadlamudi. Novel ERX-11 and CDK4/6 inhibitor combination therapy for treating therapy resistant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4148. doi:10.1158/1538-7445.AM2017-4148

Abstract B08: ESR1 coregulator binding site inhibitors (ECBIs) as novel therapeutics to target hormone therapy-resistant breast cancer

Estrogens contribute to the progression of breast cancer via estrogen receptor 1 (ESR1) and current therapies involve either antiestrogens (AE) or aromatase inhibitors (AI). However, most patients develop resistance to these drugs. Critically, therapy-resistant tumors retain ESR1-signaling. Mechanisms of therapy resistance involve the activation of ESR1 in the absence of ligand or mutations in ESR1 that allow interaction between the ESR1 and coregulators leading to sustained ESR1 signaling and proliferation. For patients with therapy-resistant breast cancers, there is a critical unmet need for novel agents to disrupt ESR1 signaling by blocking ESR1 interactions with its coregulators. Methods: Using rational design, we synthesized and evaluated a small organic molecule (ESR1 coregulator binding inhibitor, ECBI) that mimics the ESR1 coregulator nuclear receptor box motif. Using in vitro cell proliferation and apoptosis assays, we tested the effect of ECBI on several breast cancer cells and therapy-resistant model cells. Mechanistic studies were conducted using established biochemical assays, reporter gene assays, RTqPCR and RNASeq analysis. Gene differential expression lists were analyzed using Ingenuity Pathway Analysis (IPA). ESR1+ve (MCF7 and ZR75) xenografts were used for preclinical evaluation and toxicity. The efficacy of ECBI was tested using an ex vivo cultures of freshly extirpated prrimary human breast tissues. Results: In estrogen induced proliferation assays using several ESR1+ve model cells, we found that ECBI inhibit growth (IC50=300-500 nM). Importantly, ECBI showed little or no activity on ESR1 negative cells. Further, ECBI also reduced the proliferation of several ESR1 positive hormonal therapy resistant cells, directly interacted with MT-ESR1 with high affinity and significantly inhibited MT-ESR1 driven oncogenic activity. Mechanistic studies showed that ECBI interacts with ESR1, efficiently blocks ESR1 interactions with coregulators and reduces the ESR1 reporter gene activity. RNA sequencing analysis revealed that ECBI blocks multiple ESR1 driven pathways, likely representing the ability of a single ECBI compound to block multiple ESR1-coregulator interactions. Treatment of ESR1-positive xenograft tumors with ECBI (10 mg/Kg/oral) reduced tumor volume by 67% compared to control. Further, ECBI also significantly reduced the proliferation of coregulator-overexpressed breast cancer cells in xenograft model. Using human primary breast tissue ex vivo cultures, we have provided evidence that ECBI has potential to dramatically reduce proliferation of human breast tumor cells. Conclusions: The ECBI is a novel agent that targets ESR1 with a unique mechanism of action. ECBI has distinct pharmacologic advantages of oral bioavailability, in vivo stability, and is associated with minimal systemic side effects. Remarkably, ECBIs block both native and mutant forms of ESR1 and have activity against therapy resistant breast cancer cell proliferation both in vitro and in vivo and against primary human tissues ex vivo. Thus development of ECBI represents a quantum leap in therapies to target ESR1 Citation Format: Ratna K. Vadlamudi, Gangadhara Reddy Sareddy, Suryavathi Viswanadhapalli, Tae-Kyung Lee, Shi-Hong Ma, Wan Ru Lee, Monica Mann, Samaya Rajeshwari Krishnan, Vijay Gonugunta, Douglas W. Strand, Rajeshwar Rao Tekmal, JungMo Ahn, Ganesh V. Raj. ESR1 coregulator binding site inhibitors (ECBIs) as novel therapeutics to target hormone therapy-resistant breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B08.

Abstract 860: ESR1 coregulator binding inhibitor (ECBI): a novel agent for treating hormone therapy-resistant breast cancer

Background: Estrogen contribute to the progression of breast cancer via estrogen receptor 1 (ESR1) and current therapies involve either antiestrogens (AE) or aromatase inhibitors (AI). However, most patients develop resistance to these drugs. In resistant tumors, activation of ESR1 in the absence of ligand or mutations in ESR1 allow interaction between the ESR1 and coregulators leading to sustained ESR1 signaling and proliferation. Here we, developed a novel ESR1 coregulator binding inhibitor (ECBI) that targets persistent ESR1 signaling that commonly occur in therapy resistant breast tumors. Methods: Using rational design, we synthesized and evaluated a small organic molecule (ECBI) that mimics the ESR1 coregulator nuclear receptor box motif. Mechanistic studies were conducted using reporter gene assays, RT-qPCR., ChIP, and RNA-Seq analysis. Xenografts and patient derived tumors were used for preclinical evaluation and toxicity. Results: In estrogen induced proliferation assays using several ESR1+ve model cells, ECBI significantly inhibited growth and promoted apoptosis. Importantly, ECBI showed little or no activity on ESR1 negative cells. Further, ECBI also reduced the proliferation of several ESR1 positive hormonal therapy resistant cells. Mechanistic studies showed that ECBI interacts with ESR1, efficiently blocks ESR1 interactions with coregulators and reduces the ESR1 driven ERE reporter gene activity. Further, ECBI directly interacted with mutant-ESR1 with high affinity and significantly inhibited mutant-ESR1 driven oncogenic activity. RNA sequencing analysis revealed that ECBI blocks multiple ESR1 driven pathways, likely representing the ability of a single ECBI compound to block multiple ESR1-coregulator interactions. Treatment of ESR1-positive and therapy resistant as well as syngeneic xenograft tumors with ECBI (10 mg/kg/day/oral) significantly reduced the tumor volume compared to control. Using human primary breast tissue ex vivo cultures, we have provided evidence that ECBI has potential to dramatically reduce proliferation of human breast tumors. Conclusions: The ECBI is a novel agent that targets ESR1 with a unique mechanism of action. ECBI has distinct pharmacologic advantages of oral bioavailability, in vivo stability, and is associated with minimal systemic side effects. Remarkably, ECBI block both native and mutant forms of ESR1 and have activity against therapy resistant breast cancer cell proliferation both in vitro and in vivo and against primary human tumor tissues ex vivo. This first-in-class agent with its novel mechanism of action overcomes the limitations of current therapies. Citation Format: Ratna K. Vadlamudi, Gangadhara Reddy Sareddy, Suryavathi Viswanadhapalli, Tae-Kyung Lee, Shi-Hong Ma, Wan Ru Lee, Monica Mann, Samaya Rajeshwari Krishnan, Vijay Gonugunta, Yang Liu, Douglas W. Strand, Rajeshwar Rao Tekmal, Jung-Mo Ahn, Ganesh V. Raj. ESR1 coregulator binding inhibitor (ECBI): a novel agent for treating hormone therapy-resistant breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 860.

Multiple Labeling of Peptides via Orthogonal Coupling Reactions and Its Applications

Labeling peptides have been widely practiced in numerous applications in chemistry and biology. For example, fluorescent dyes make peptides useful probes for in vitro and in vivo imaging and suitable substrates for determining enzyme activity; biotinylation has vast applications including affinity-based protein purification; PEGylation enhances plasma half-life and protects against metabolic degradation; radiolabeling offers in vitro and in vivo molecular imaging agents and targeted radionuclide therapy; cell permeable peptides give access to intracellular targets. Thus, peptides bearing such multiple labels may serve as powerful research tools and become handy for diverse biomedical studies. For labeling peptides, a number of coupling methods have been reported, such as alkyne-azide click chemistry, thiol-maleimide coupling, Staudinger ligation, and oxime/hydrazone formation. Despite the versatility and broad use in traditional organic reactions, Suzuki-Miyaura coupling has been rarely practiced in peptide labeling. In addition, while each of these methods has been efficiently used alone in peptide conjugation, their combination for orthogonal coupling reactions was not well studied.