Peptidomimetic Libraries Research Articles

Smart Electrospun Nanofibers from Short Peptidomimetics Based on Pyrrolo-pyrazole Scaffold.

We prepared a small library of short peptidomimetics based on 3-pyrrolo-pyrazole carboxylate, a non-coded γ-amino acid, and glycine or alanine. The robust and eco-friendly synthetic approach adopted allows to obtain the dipeptides in two steps from commercial starting materials. This gives the possibility to shape these materials by electrospinning into micro- and nanofibers, in amounts required to be useful for coating surfaces of biomedical relevance. To promote high quality of electrospun fibers, different substitution patterns were evaluated, all for pure peptide fibers, free of any polymer or additive. The best candidate, which affords a homogeneous fibrous matrix, was prepared in larger amounts, and its biocompatibility was verified. This successful work is the first step to develop a new biomaterial able to produce pristine peptide-based nanofibers to be used as helpful component or stand-alone scaffolds for tissue engineering or for the surface modification of medical devices.

Peptidomimetics as potent dual SARS-CoV-2 cathepsin-L and main protease inhibitors: In silico design, synthesis and pharmacological characterization

In this paper we present the design, synthesis, and biological evaluation of a new series of peptidomimetics acting as potent anti-SARS-CoV-2 agents. Starting from our previously described Main Protease (MPro) and Papain Like Protease (PLPro) dual inhibitor, CV11, here we disclose its high inhibitory activity against cathepsin L (CTSL) (IC50 = 19.80 ± 4.44 nM), an emerging target in SARS-CoV-2 infection machinery. An in silico design, inspired by the structure of CV11, led to the development of a library of peptidomimetics showing interesting activities against CTSL and Mpro, allowing us to trace the chemical requirements for the binding to both enzymes. The screening in Vero cells infected with 5 different SARS-CoV-2 variants of concerns, highlighted sub-micromolar activities for most of the synthesized compounds (13, 15, 16, 17 and 31) in agreement with the enzymatic inhibition assays results. The compounds showed lack of activity against several different RNA viruses except for the 229E and OC43 human coronavirus strains, also characterized by a cathepsin-L dependent release into the host cells. The most promising derivatives were also evaluated for their chemical and metabolic in-vitro stability, with derivatives 15 and 17 showing a suitable profile for further preclinical characterization.

Synthesis, derivatization, and conformational scanning of peptides containing N-Aminoglycine.

N-alkylated glycine residues are the main constituent of peptoids and peptoid-peptide hybrids that are employed across the biomedical and materials sciences. While the impact of backbone N-alkylation on peptide conformation has been extensively studied, less is known about the effect of N-amination on the secondary structure propensity of glycine. Here, we describe a convenient protocol for the incorporation of N-aminoglycine into host peptides on solid support. Amide-to-hydrazide substitution also affords a nucleophilic handle for further derivatization of the backbone. To demonstrate the utility of late-stage hydrazide modification, we synthesized and evaluated the stability of polyproline II helix and β-hairpin model systems harboring N-aminoglycine derivatives. The described procedures provide facile entry into peptidomimetic libraries for conformational scanning.

Advances in Ultrahigh Throughput Hit Discovery with Tandem Mass Spectrometry Encoded Libraries.

Discovering new bioactive molecules is crucial for drug development. Finding a hit compound for a new drug target usually requires screening of millions of molecules. Affinity selection based technologies have revolutionized early hit discovery by enabling the rapid screening of libraries with millions or billions of compounds in short timeframes. In this Perspective, we describe recent technology breakthroughs that enable the screening of ultralarge synthetic peptidomimetic libraries with a barcode-free tandem mass spectrometry decoding strategy. A combination of combinatorial synthesis, affinity selection, automated de novo peptide sequencing algorithms, and advances in mass spectrometry instrumentation now enables hit discovery from synthetic libraries with over 100 million members. We provide a perspective on this powerful technology and showcase success stories featuring the discovery of high affinity binders for a number of drug targets including proteins, nucleic acids, and specific cell types. Further, we show the usage of the technology to discover synthetic peptidomimetics with specific functions and reactivity. We predict that affinity selection coupled with tandem mass spectrometry and automated de novo decoding will rapidly evolve further and become a broadly used drug discovery technology.

Expanding the therapeutic options for Candida infections using novel inhibitors of secreted aspartyl proteases.

For widening the therapeutic options for Candida management, the druggability of Candida proteome was systematically investigated using an innovative pipeline of high-throughput data mining algorithms, followed by in vitro validation of the observations. Through this exercise, HIV-1 protease was found to share structural similarity with secreted aspartyl protease-3 (SAP3), a virulence protein of Candida. Using the molecular fingerprint of HIV-1 protease inhibitor GRL-09510, we performed virtual screening of peptidomimetic library followed by high-precision docking and MD simulations for discovery of SAP inhibitors. Wet-lab validation of the four shortlisted peptidomimetics revealed that two molecules, when used in combination with fluconazole, could significantly reduce the dosage of fluconazole required for 50% inhibition of Candida albicans. The SAP inhibitory activity of these peptidomimetics was confirmed through SAP assays and found to be on par with pepstatin A, a known peptidomimetic inhibitor of aspartyl proteases.

Peptidomimetics designed to bind to RAS effector domain are promising cancer therapeutic compounds

Oncogenic RAS proteins are important for driving tumour formation, and for maintenance of the transformed phenotype, and thus their relevance as a cancer therapeutic target is undeniable. We focused here on obtaining peptidomimetics, which have good pharmacological properties, to block Ras–effector interaction. Computational analysis was used to identify hot spots of RAS relevant for these interactions and to screen a library of peptidomimetics. Nine compounds were synthesized and assayed for their activity as RAS inhibitors in cultured cells. Most of them induced a reduction in ERK and AKT activation by EGF, a marker of RAS activity. The most potent inhibitor disrupted Raf and PI3K interaction with oncogenic KRAS, corroborating its mechanism of action as an inhibitor of protein–protein interactions, and thus validating our computational methodology. Most interestingly, improvement of one of the compounds allowed us to obtain a peptidomimetic that decreased the survival of pancreatic cancer cell lines harbouring oncogenic KRAS.

Mapping of FSHR agonists and antagonists binding sites to identify potential peptidomimetic modulators.

Owing to the critical role of follicle stimulating hormone receptor (FSHR) signaling in human reproduction, FSHR has been widely explored for development of fertility regulators. Using high-throughput screening approaches, several low molecular weight (LMW) compounds that can modulate FSHR activity have been identified. However, the information about the binding sites of these molecules on FSHR is not known. In the present study, we extracted the structural and functional information of 161 experimentally validated LMW FSHR modulators available in PubMed records. The potential FSHR binding sites for these modulators were identified through molecular docking experiments. The binding sites were further mapped to the agonist or antagonist activity reported for these molecules in literature. MD simulations were performed to evaluate the effect of ligand binding on conformational changes in the receptor, specifically the transmembrane domain. A peptidomimetic library was screened using these binding sites. Six peptidomimetics that interacted with the residues of transmembrane domain and extracellular loops were evaluated for binding activity using in vitro cAMP assay. Two of the six peptidomimetics exhibited positive allosteric modulatory activity and four peptidomimetics exhibited negative allosteric modulatory activity. All six peptidomimetics interacted with Asp521 of hFSHR(TMD). Several of the experimentally known LMW FSHR modulators also participated in H-bond interactions with Asp521, suggesting its important role in FSHR modulatory activity.

Rapid de novo discovery of peptidomimetic affinity reagents for human angiotensin converting enzyme 2

Rapid discovery and development of serum-stable, selective, and high affinity peptide-based binders to protein targets are challenging. Angiotensin converting enzyme 2 (ACE2) has recently been identified as a cardiovascular disease biomarker and the primary receptor utilized by the severe acute respiratory syndrome coronavirus 2. In this study, we report the discovery of high affinity peptidomimetic binders to ACE2 via affinity selection-mass spectrometry (AS-MS). Multiple high affinity ACE2-binding peptides (ABP) were identified by selection from canonical and noncanonical peptidomimetic libraries containing 200 million members (dissociation constant, KD = 19–123 nM). The most potent noncanonical ACE2 peptide binder, ABP N1 (KD = 19 nM), showed enhanced serum stability in comparison with the most potent canonical binder, ABP C7 (KD = 26 nM). Picomolar to low nanomolar ACE2 concentrations in human serum were detected selectively using ABP N1 in an enzyme-linked immunosorbent assay. The discovery of serum-stable noncanonical peptidomimetics like ABP N1 from a single-pass selection demonstrates the utility of advanced AS-MS for accelerated development of affinity reagents to protein targets.

One Bead-One Compound (OBOC) Peptidomimetic-Encoded Library Synthesis via Split-and-Pool Methods.

Large structurally diverse peptidomimetic chemical libraries have been very useful tools in chemical biology and drug discovery for the identification of therapeutically important compounds with higher affinity and improved pharmacological properties against different protein targets.Here we describe a simple and general method for the submonomer solid phase synthesis of large one bead-one compound (OBOC) peptidomimetic libraries of structurally diverse compounds that can be encoded by mass or genetic methods.

The Synthesis and Evaluation of Aminocoumarin Peptidomimetics as Cytotoxic Agents on Model Bacterial E. coli Strains.

This work presents the successful synthesis of a library of novel peptidomimetics via Ugi multicomponent reaction. Most of these peptidomimetics contain differently substituted aminocoumarin; 7-amino-4-methylcoumarin and 7-amino-4-(trifluoromethyl) coumarin. Inspired by the biological properties of coumarin derivatives and peptidomimetics, we proposed the synthesis of coumarin incorporated peptidomimetics. We studied the potential of synthesized compounds as antimicrobial drugs on model E. coli bacterial strains (k12 and R2–R4). To highlight the importance of coumarin in antimicrobial resistance, we also synthesized the structurally similar peptidomimetics, using benzylamine. Preliminary cellular studies suggest that the compounds with coumarin derivatives have more potential as antimicrobial agents compared to the compounds without coumarin. We also analyzed the effect of aldehyde, free acid group and ester group on the course of their antimicrobial properties.

Polyphenylglyoxamide-Based Amphiphilic Small Molecular Peptidomimetics as Antibacterial Agents with Anti-Biofilm Activity.

The rapid emergence of drug-resistant bacteria is a major global health concern. Antimicrobial peptides (AMPs) and peptidomimetics have arisen as a new class of antibacterial agents in recent years in an attempt to overcome antibiotic resistance. A library of phenylglyoxamide-based small molecular peptidomimetics was synthesised by incorporating an N-alkylsulfonyl hydrophobic group with varying alkyl chain lengths and a hydrophilic cationic group into a glyoxamide core appended to phenyl ring systems. The quaternary ammonium iodide salts 16d and 17c showed excellent minimum inhibitory concentration (MIC) of 4 and 8 μM (2.9 and 5.6 μg/mL) against Staphylococcus aureus, respectively, while the guanidinium hydrochloride salt 34a showed an MIC of 16 μM (8.5 μg/mL) against Escherichia coli. Additionally, the quaternary ammonium iodide salt 17c inhibited 70% S. aureus biofilm formation at 16 μM. It also disrupted 44% of pre-established S. aureus biofilms at 32 μM and 28% of pre-established E. coli biofilms 64 μM, respectively. A cytoplasmic membrane permeability study indicated that the synthesised peptidomimetics acted via disruption and depolarisation of membranes. Moreover, the quaternary ammonium iodide salts 16d and 17c were non-toxic against human cells at their therapeutic dosages against S. aureus.

Peptide scaffold‐derived peptidomimetic farnesyltransferase inhibitors

AbstractHuman protein farnesyltransferase (FTase) is involved in many essential signal transduction proteins and has been explored as druggable targets of diverse diseases such as cancer and inflammation. Here, we propose a new strategy termed as Peptide Scaffold‐derived Peptidomimetic FTase Inhibitors (PSPFI) to discover peptidomimetic lead molecular entities that can inhibit FTase. The strategy first defines a PSPFI‐oriented peptidomimetic library that contains >30,000 sulfhydryl/thioether‐carrying peptidomimetics, and then employs molecular docking calculation, multiple similarity analysis, structural minimization, and affinity scoring to screen against the library, in order to discover those peptidomimetics that share high similarity with the core binding module (CBM) of FTase Rap2a peptide substrate in 2D‐chemistry, 3D‐conformation, pharmacophore, and nonbonded pattern, as well as exhibit high theoretical affinity to FTase. Totally 56 hit peptidomimetics were identified from the library, from which we manually select 6 structurally diverse, purchasable peptidomimetics to perform FTase assays. Consequently, four are determined as potent FTase inhibitors (IC50 < 1 μM); their inhibitory activity is moderately lower than the sophisticated FTase inhibitor Tipifarnib and considerably higher than the native CBM peptide. These peptidomimetic ligands share similar extended conformation, binding mode, and Zn2+ coordination with Rap2a CBM peptide in FTase active site, but would have a different inhibition mechanism to Tipifarnib.

On-resin multicomponent protocols for biopolymer assembly and derivatization.

Solid-phase synthesis represents the methodological showcase for technological advances such as split-and-pool combinatorial chemistry and the automated synthesis of peptides, nucleic acids and polysaccharides. These strategies involve iterative coupling cycles that do not generate functional diversity besides that incorporated by the amino acids, nucleosides and monosaccharide building blocks. In sharp contrast, multicomponent reactions (MCRs) are traditionally used to generate both skeletal and appendage diversity in short, batchwise procedures. On-resin MCRs have traditionally been employed for the construction of heterocycle and peptidomimetic libraries, but that scenario has changed recently, and today the focus is more on the solid-phase derivatization of peptides and oligonucleotides. This review presents relevant experimental details and addresses the synthetic scope of such on-resin multicomponent protocols employed to accomplish specific biopolymer covalent modifications that are practically inviable by traditional solution-phase methodologies. Recommendations are provided to facilitate the implementation of solid-supported protocols and avoid possible pitfalls associated with the selection of the polymeric resin, the solvent and the order and amount of the reagents employed. We describe procedures comprising the multicomponent lipidation, biotinylation and labeling of both termini and the side chains, as well as the use of MCRs in the traceless on-resin synthesis of ligated and cyclic peptides. Solid-phase protocols for the assembly of α-helical and parallel β-sheet peptides as well as hybrid peptide-peptoid and peptide-peptide nucleic acid architectures are described. Finally, the solid-supported multicomponent derivatization of DNA oligonucleotides is illustrated as part of the DNA-encoded library technology relying on MCR-derived heterocyclic compounds.

Automated affinity selection for rapid discovery of peptide binders.

In-solution affinity selection (AS) of large synthetic peptide libraries affords identification of binders to protein targets through access to an expanded chemical space. Standard affinity selection methods, however, can be time-consuming, low-throughput, or provide hits that display low selectivity to the target. Here we report an automated bio-layer interferometry (BLI)-assisted affinity selection platform. When coupled with tandem mass spectrometry (MS), this method enables both rapid de novo discovery and affinity maturation of known peptide binders with high selectivity. The BLI-assisted AS-MS technology also features real-time monitoring of the peptide binding during the library selection process, a feature unattainable by current selection approaches. We show the utility of the BLI AS-MS platform toward rapid identification of novel nanomolar (dissociation constant, KD < 50 nM) non-canonical binders to the leukemia-associated oncogenic protein menin. To our knowledge, this is the first application of BLI to the affinity selection of synthetic peptide libraries. We believe our approach can significantly accelerate the use of synthetic peptidomimetic libraries in drug discovery.

Validation of ligands targeting metacaspase-2 (MCA2) from Trypanosoma brucei brucei and their application to MCA5 from T. congolense as possible trypanocides

Metacaspases (MCAs) are ideal drug and diagnostic targets for animal and human African trypanosomiasis, as these cysteine peptidases are absent from the metazoan kingdom and have been implicated in the parasite cell cycle and cell death. Tsetse fly-transmitted trypanosomes that live free in the bloodstream and/or cerebrospinal fluid of the mammalian host cause animal and human African trypanosomiasis (nagana or sleeping sickness respectively). Chemotherapy and chemoprophylaxis are the main forms of control, but in contrast to human trypanocides, the veterinary drugs are old and drug resistance is on the increase. A peptidomimetic library targeting the MCA2 from Trypanosoma brucei brucei has ligands with low IC50 values, some of which were antiparasitic. This study validates the inhibitory activity of these ligands using the protein structure solved by X-ray diffraction after the ligand library was published. Water molecules were shown to be important in substrate binding and strategies to improve the efficacy of these ligands are highlighted. These ligands appear to be pan-specific as they were docked into the active site of the homology modelled MCA5 of animal infective Trypanosoma congolense with similar binding energies and conformations.

Abstract P3-10-01: Development and characterization of a first-in-class small molecule inhibitor of PELP1

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.

\u03b3-AApeptides\u2013based Small Molecule Ligands That Disaggregate Human Islet Amyloid Polypeptide

The abnormal folding and aggregation of functional proteins into amyloid is a typical feature of many age-related diseases, including Type II diabetes. Growing evidence has revealed that the prevention of aggregate formation in culprit proteins could retard the progression of amyloid diseases. Human Amylin, also known as human islet amyloid polypeptide (hIAPP), is the major factor for categorizing Type II diabetes as an amyloid disease. Specifically, hIAPP has a great aggregation potential, which always results in a lethal situation for the pancreas. Many peptide inhibitors have been constructed from the various segments of the full-length hIAPP peptide; however, only a few have their origin from the screening of combinatorial peptidomimetic library. In this study, based on HW-155, which was previously discovered from a one–bead–one compound (OBOC) library to inhibit Aβ40 aggregation, we investigated eight (8) analogues and evaluated their amyloid-prevention capabilities for inhibiting fibrillization of hIAPP. Characterization studies revealed that all analogues of HW-155, as well as HW-155, were effective inhibitors of the fibril formation by hIAPP.

Screening of Chemical Libraries Using a Yeast Model of Retinal Disease

Retinitis pigmentosa (RP) is a degenerative retinal disease, often caused by mutations in the G-protein-coupled receptor rhodopsin. The majority of pathogenic rhodopsin mutations cause rhodopsin to misfold, including P23H, disrupting its crucial ability to respond to light. Previous screens to discover pharmacological chaperones of rhodopsin have primarily been based on rescuing rhodopsin trafficking and localization to the plasma membrane. Here, we present methods utilizing a yeast-based assay to screen for compounds that rescue the ability of rhodopsin to activate an associated downstream G-protein signaling cascade. We engineered a yeast strain in which human rhodopsin variants were genomically integrated, and were able to demonstrate functional coupling to the yeast mating pathway, leading to fluorescent protein expression. We confirmed that a known pharmacological chaperone, 9-cis retinal, could partially rescue light-dependent activation of a disease-associated rhodopsin mutation (P23H) expressed in yeast. These novel yeast strains were used to perform a phenotypic screen of 4280 compounds from the LOPAC1280 library and a peptidomimetic library, to discover novel pharmacological chaperones of rhodopsin. The fluorescence-based assay was robust in a 96-well format, with a Z′ factor of 0.65 and a signal-to-background ratio of above 14. One compound was selected for additional analysis, but it did not appear to rescue rhodopsin function in yeast. The methods presented here are amenable to future screens of small-molecule libraries, as they are robust and cost-effective. We also discuss how these methods could be further modified or adapted to perform screens of more compounds in the future.

Abstract P4-07-01: A small molecule inhibitor (ERX-41) induces endoplasmic reticulum stress in triple negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer and represents a disproportional share of the breast cancer mortality, primarily due to a lack of targeted therapies. There is a major unmet need for rationally designed novel therapies that can extend survival of patients with TNBC. TNBCs are characterized by a high basal level of endoplasmic reticulum stress, due to high protein turnover and need for proliferation. Recent studies revealed the role of several members of the Nuclear Receptor (NR) superfamily as molecular drivers in TNBC, including the androgen receptor (AR), glucocorticoid receptor (GR) and the orphan NR tailless (TLX). Methods: Recently, using peptidomimetics, we have developed small molecules that specifically target and block interactions of multiple coregulators with oncogenic NRs. We performed a screen of our 500+ compound peptidomimetic library derived from our ERX-11 oligobenzamide (that was rationally designed to target ERα) for anti-proliferative activity in TNBC cell lines. Identified leads were then validated in multiple TNBC cell lines. In vitro activity was tested using Cell titer glo, MTT, matrigel invasion, and apoptosis assays. Mechanistic studies were conducted using Western blot, reporter gene assays, CRISPR/Cas9 KO and RNA-seq analysis. Xenograft, patient derived xenograft (PDX), patient derived explant (PDE) and xenograft derived explant (XDE) TNBC models were used for preclinical evaluation and toxicity. Results: We have identified a first-in-class drug (ERX-41) that has potent activity (IC50 = 50-250nM) against all six molecular subtypes of TNBC. Systematic evaluation using CRISPR/Cas9 KO screen and overexpression screen comprising 48 NRs identified TLX as a preferred target of ERX-41. Analyses of primary breast tumors revealed TLX was highly expressed in TNBC. Further, TLX was amplified in nearly 50% of TNBC xenografts (cbioportal.org). Modelling, mechanistic and biochemical studies showed that ERX-41 interact with TLX and selectively blocks its interactions with coregulators. Gene expression analyses revealed both significant reduction of TLX-activated genes (CCND1, WNT7A) and significant activation of TLX-repressed genes (p21) upon treatment with ERX-41 in TNBC models. Gene ontogeny pathway analyses of RNA-seq data in TNBC cells showed that ERX-41 treatment positively correlated with apoptosis. Our ultrastructural studies indicated that ERX-41 enhances endoplasmic reticulum stress in TNBC inducing autophagic flux and subsequent apoptosis. ERX-41 has significant potency against multiple TNBC xenografts and PDXs in vivo, PDEs and XDEs ex vivo, indicating its potential for clinical translation. Pharmacologically, ERX-41 exhibited high oral bioavailability and associated with minimal toxicity upon oral gavage for up to 120 days in animal studies. Conclusions: We believe that the ability of ERX-41 to block NR signaling and target a critical molecular vulnerability in TNBC and its ability to enhance endoplasmic reticulum stress in TNBC, will revolutionize the therapeutic landscape of TNBC. ERX-41 is oral bioavailable, potent against multiple TNBC molecular subtypes, and is associated with minimal systemic side effects. (supported by NIH grant RO1 CA223828-01) Citation Format: Liu X, Viswanadhapalli S, Ma S, Lee T-K, Sareddy GR, Ekoue DN, Blatt EM, Zhou M, Li M, Tekmal RR, Ahn J-m, Vadlamudi RK, Raj GV. A small molecule inhibitor (ERX-41) induces endoplasmic reticulum stress in triple negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-07-01.

Cyclic Peptidomimetics as Inhibitor for miR-155 Biogenesis.

miR-155 plays key promoting roles in several cancers and emerges as an important anticancer therapeutic target. However, the discovery of small molecules that target RNAs is challenging. Peptidomimetics have been shown to be a rich source for discovering novel ligands to regulate cellular proteins. However, the potential of using peptidomimetics for RNA targeting is relatively unexplored. To this end, we designed and synthesized members of a novel 320 000 compound macrocyclic peptidomimetic library. An affinity-based screening protocol led to the identification of a pre-miR-155 binder that inhibits oncogenic miR-155 maturation in vitro and in cell and induces cancer cell apoptosis. The results of this investigation demonstrate that macrocyclic peptidomimetics could serve as a new scaffold for RNA targeting.