Abstract P3-10-01: Development and characterization of a first-in-class small molecule inhibitor of PELP1
Authors: Altwegg Kristin A , Viswanadhapalli Suryavathi , Mann Monica , Pratap Uday P , Li Mengxing , Liu Junhao , Luo Yiliao , Sareddy Gangadhara R , Vankayalapati Hariprasad , Vadlamudi Ratna K. 
Published: March 2020

Abstract
Background: Breast cancer (BCa) is the most commonly diagnosed cancer and the second leading cause of cancer death in women. BCa is composed of distinct molecular subtypes, such as ER positive BCa (ER+ BCa) and triple negative BCa (TNBC). Development of novel effective therapies for patients with therapy resistant breast cancer (TR-BC) and TNBC remains the highest unmet need in patient treatment and survivorship. Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) plays a critical role in multiple nuclear receptor functions leading to TR-BC and TNBC progression. PELP1 expression is dysregulated in BCa, is a prognostic indicator of poorer BCa survival, and its deregulation contributes to BCa therapy resistance. The objective of this study is development and characterization of a small molecule inhibitor of PELP1 (SMIP) as novel therapeutic for treating BCa.
Methods: Using yeast two-hybrid screening, we identified PELP1 Inhibitory Peptide (PIP1) from a library of peptides. PIP1 binds PELP1 with high affinity and functions to inhibit PELP1 oncogenic activity. Direct binding of PIP1 to PELP1 was confirmed using biotin pull-down assays and inhibition of BCa proliferation confirmed using MTT assays. We used the Hit-Ligand interaction site with PIP1 hot spot residues based on 3D alignment and morphology to generate a library of peptidomimetics (small chemical molecules). In vitro activity was assessed using Celltiter Glo, MTT, and matrigel invasion chamber assays in multiple BCa models. Mechanistic studies were conducted using Western blot, reporter gene assays, and peptide competition assays. Xenograft and patient derived explant (PDEX) assays were used for preclinical evaluation and preliminary toxicity analysis.
Results: Bioactivity screens revealed PELP1 Inhibitory Peptide (PIP1) significantly attenuates PELP1-mediated proliferation with an IC50 of 10µM across multiple BCa cell lines. We confirmed PIP1 binding to PELP1 using peptide pull-down assays with nuclear lysates from BCa cells. Using Hit-Ligand-Based interaction site with the PIP1 hot spot residues, we identified 61 potential hits using a 10,000 Diverse Set. Screening of these 61 potential hits using the MTT assays lead to the selection of SMIP34 (tetrahydropyrazolo [1,5a) pyrazole) as lead inhibitor of PELP1. SMIP34 treatment reduced proliferation at an IC50 of 3-10µM in ER+ BCa (ZR-75, MCF-7, and T- 47D); TR-BC (MCF-7-TamR, MCF-7-LTLT, ZR-75-MT-ER537S, and ZR-75-MT-ER538G); and TNBC (MDA- MB-231, and BT549) models. Additionally, SMIP34 showed no activity in human mammary epithelial cells. Specificity of SMIP34 was confirmed using PELP1 knockdown BCa cell lines. Mechanistic studies using Western blot analysis confirmed that SMIP34 binding to PELP1 contributes to its degradation. In matrigel invasion chamber assays, SMIP34 significantly reduced the invasiveness of TR-BC and TNBC models. In combination studies, SMIP34 displayed synergy and enhanced the efficacy of current chemotherapeutics Cisplatin and Paclitaxel. In PDEX assays, Ki67 staining revealed SMIP34 significantly decreased tumor proliferation. In xenograft models, SMIP34 (10mg/kg/s.c.) treatment resulted in significant reduction in tumors compared to vehicle treatment. Furthermore, overall mouse body weight in both control and SMIP34 treated groups were similar, suggesting no overt signs of toxicity.
Conclusion: We have developed a first-in-class small molecule inhibitor of PELP1 (SMIP) displaying effectivity against TR-BC and TNBC in vitro and in vivo.
Supported by CPRIT Predoctoral Fellowship CPRIT RTA; RP170345 (K.A. Altwegg)
Citation Format: Kristin A Altwegg, Suryavathi Viswanadhapalli, Monica Mann, Uday P Pratap, Mengxing Li, Junhao Liu, Yiliao Luo, Gangadhara R Sareddy, Hariprasad Vankayalapati, Ratna K. Vadlamudi. Development and characterization of a first-in-class small molecule inhibitor of PELP1 [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-10-01.