Small Molecule Microarray Screen Research Articles

Abstract A057: Small molecule microarray screening identifies novel androgen receptor ligands

Abstract The androgen receptor (AR) is a well-established oncogenic driver of prostate cancer, and AR-targeting hormone therapy has proven an efficacious treatment option for patients with metastatic prostate cancer. Due to the importance of AR in prostate cancer disease progression, we leveraged small molecule microarray (SMM) screening technology to identify compounds that bind to transcriptional complexes of a common AR splice-variant, AR-V7, present along with AR in castration-resistant prostate cancer cell lysates. A subset of SMM hits were identified to modulate the AR Transcription Regulatory Network (TRN) in hormone-sensitive, but not castration-resistant prostate cancer models using a functional cell-based transcription signature approach. Several AR SMM hits directly engaged the AR ligand binding domain in a biophysical assay. One of these AR SMM hits altered AR nuclear translocation and antagonized steroid receptor co-activator recruitment to AR but did not antagonize the glucocorticoid receptor. Leveraging structural data for known AR ligands, we propose a computational model for binding of this molecule to the known AR ligand binding domain. Together, the data demonstrates that the AR SMM lysate screen successfully identified structurally novel small molecules that modulate the AR TRN and bind the AR ligand binding domain. Citation Format: Marek J Kobylarz, Michelle G Shum, Wayne L Glore, Ava Saffren, Shannon Carpenter, Mulini Pingili, Andrew Clay, Tamara D Hopkins, Hua Gao, Anna C Schinzel, Marius S Pop, Nikolaus D Obholzer, David B Freeman, Christopher J Dinsmore, Benjamin W Trotter, Charles Y Lin, Peter B Rahl. Small molecule microarray screening identifies novel androgen receptor ligands [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A057.

Investigating the NRAS 5′ UTR as a target for small molecules

Neuroblastoma RAS (NRAS) is an oncogene that is deregulated and highly mutated in cancers including melanomas and acute myeloid leukemias. The 5' untranslated region (UTR) (5' UTR) of the NRAS mRNA contains a G-quadruplex (G4) that regulates translation. Here we report a novel class of small molecule that binds to the G4 structure located in the 5' UTR of the NRAS mRNA. We used a small molecule microarray screen to identify molecules that selectively bind to the NRAS-G4 with submicromolar affinity. One compound inhibits the translation of NRAS invitro but showed only moderate effects on the NRAS levels in cellulo. Rapid Amplification of cDNA Ends and RT-PCR analysis revealed that the predominant NRAS transcript does not possess the G4 structure. Thus, although NRAS transcripts lack a G4 in many cell lines the concept of targeting folded regions within 5' UTRs to control translation remains a highly attractive strategy.

The binding of LDN193189 to CD133 C-terminus suppresses the tumorigenesis and immune escape of liver tumor-initiating cells

The tumor-initiating cell (TIC) marker CD133 promotes TIC self-renewal and tumorigenesis through the tyrosine phosphorylation of its c-terminal domain. Therefore, finding compounds that target the phosphorylation of CD133 will provide an effective method for inhibiting TICs characteristics. Here, through small molecule microarray screening, compound LDN193189 was found to bind to the c-terminus of CD133 and influenced its tyrosine phosphorylation. LDN193189 inhibited the interaction between CD133 and p85, accompanied by a reduction in the self-renewal and tumorigenicity of liver TIC. In addition, LDN193189 inhibited the expression and transcription of Galectin-3 by reducing the tyrosine phosphorylation of CD133. Galectin-3 secreted by liver TICs inhibited the proliferation of activated CD8+ T cells by binding to PD-1. LDN193189 suppressed the immune escape ability of liver TICs by downregulating Galectin-3. Taken together, LDN193189 suppressed the tumorigenesis and immune escape of liver CSCs by targeting the CD133-Galectin-3 axis.

Max-imizing the Attenuation of Myc Using Small Molecules

It has been a widely held notion within the biomedical research community that the reliable modulation of transcription factors with small molecules would represent a holy grail, given their role in directly potentiating oncogenic programs. Among the transcription factors that have been held in highest regard is Myc, since its dysregulation is among the most recurrent events in human cancer. Despite intense efforts, the ability to identify compounds that bind directly to Myc, resulting in its functional inhibition, have been met with only moderate success. However, a new approach reported by Struntz et al. (Cell Chem. Biol., 2019) focuses on a different strategy of discovering molecules that bind to Myc's obligate partner Max. Using a small-molecule microarray screen, they report the identification of KI-MS2-008, a compound that results in the stabilization of Max homodimers and the attenuation of Myc. KI-MS2-008 suppresses cancer cell grown both in vitro and within in vivo models.

An Array-Based Ligand Discovery Platform for Proteins With Short Half-Lives.

Many promising therapeutic protein targets were previously considered "undruggable" due to a deficit in structural information to guide drug design and/or a lack of an obvious binding pocket. Fortunately, array-based methods for evaluating protein binding against large chemical libraries, such as small-molecule microarray screening, have provided one of several emerging inroads to ligand discovery for these elusive targets. Despite the advance in the area of ligand discovery for poorly structured and intrinsically disordered proteins provided by array-based technologies involving cell lysates, the extension of this technology for screening proteins with short half-lives in physiologically relevant conformations has been technically challenging. In this chapter we present a protocol for leveraging in vitro translation strategies to enable array-based screening of short-lived proteins against large small-molecule libraries for ligand discovery.

Abstract 322: Selective inhibition of MYC expression by a small molecule G-quadruplex stabilizer

Abstract MYC overexpression or amplification is commonly seen in a wide variety of tumor types, including multiple myeloma. Although MYC appears to be an important driver of the oncogenic phenotype, the helix-loop-helix topology, short half-life, and rapid replenishment of its encoded protein make it a particularly challenging drug target. However, the promoter region of the MYC gene contains a G-quadruplex (G4) DNA secondary structure that can be stabilized to block transcription. Recently, we reported the identification of a compound from a small molecule microarray screen that selectively binds the MYC G4, compared with other oncogenes that also contain G4s, and inhibits MYC mRNA and protein expression, leading to cell cycle arrest in human multiple myeloma cell lines (ACS Chem Biol. 2015). Here we describe the in vitro and in vivo evaluation of a more potent analog that retains the selectivity of the original compound for the MYC G4 sequence. After observing strong growth inhibitory activity of this new compound in a diverse panel of human cancer cell lines, we evaluated its pharmacokinetic and toxicity profiles in mice. Preliminary pharmacokinetic studies showed promising serum bioavailability and exposure properties when administered either intravenously or intraperitoneally. The compound was well tolerated in a month long, dose-escalation toxicity study in mice receiving daily administration of the compound. No adverse effects were observed during the study. In an assessment of short term in vivo activity, MYC expression was inhibited in multiple myeloma xenografts. More long-term studies to evaluate the anti-tumor activity of the compound are currently in progress. Thus, our data provide evidence that small molecule stabilization of the MYC G4 can drive transcriptional silencing of oncogenic MYC expression both in vitro and in vivo. Citation Format: Elena C. Leon, John K. Simmons, David Calabrese, Wendy DuBois, Kenneth Felsenstein, Lindsey B. Saunders, Shuling Zhang, Aleksandra Michalowski, Frank Gonzalez, Beverly A. Mock, John S. Schneekloth. Selective inhibition of MYC expression by a small molecule G-quadruplex stabilizer. [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 322.

Abstract B40: Selective suppression of Myc transcription with a G-quadruplex stabilizing small molecule

Abstract The transcription factor Myc is disregulated in some 70% of human cancers, where it triggers aberrant growth and signaling, and contributes to tumorigenesis. Thus, Myc is a highly sought after drug target. However, targeting the Myc protein directly with small molecules has proven to be a significant challenge due to a lack of small molecule binding sites and rapid protein turnover in the cell. To date, most successes in inhibiting Myc function have come through alternative indirect approaches such as BET bromodomain inhibition. Here, we present a novel class of small molecules that silences Myc expression by stabilization of the G-quadruplex structure present in the NHEIII region of the Myc promoter. We used a small molecule microarray screen to identify molecules that selectively bind to the Myc G-quadruplex. Biophysical studies, including thermal melt and SPR analysis, demonstrated that the compound binds reversibly to the quadruplex structure with low micromolar affinity. Next, we demonstrated that the compound inhibited polymerase progression in an in vitro PCR-stop assay in a dose dependent fashion, while the compound had no effect on amplification of an oligo incapable of forming a quadruplex. Target specificity was demonstrated using a cell-based assay where one copy of the Myc gene has a translocation and is no longer under the control of a quadruplex. In this assay, Myc is only silenced upon treatment when there is a quadruplex in the promoter. Further studies demonstrate dose- and time-dependent effects on cell viability and Myc protein levels in a panel of myeloma cells. The compound also had minimal effects on viability of PBMCs from a healthy donor. Finally, gene expression analysis demonstrated that in contrast to other quadruplex-stabilizing small molecules, the compound reported here does not silence several other genes that are also under the control of quadruplexes. Citation Format: John K. Simmons, Lindsey B. Saunders, Kenneth Felsenstein, Peter Gareiss, Shuling Zhang, Beverly Mock, John S. Schneekloth, Jr.. Selective suppression of Myc transcription with a G-quadruplex stabilizing small molecule. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B40.

Small molecule microarray screening methodology based on surface plasmon resonance imaging

In order to increase the scope and utility of small molecule microarrays (SMMs) we have combined SMMs and SPRi to screen small molecule antagonists against protein targets. Several small molecules, including immunosuppressive drugs (rapamycin and FK506) and reported inhibitors (FOBISIN and Blapsin) of 14-3-3ζ proteins have been used to validate this technology. Furthermore, a small library of isatin derivatives have been synthesized and screened on developed platform against 14-3-3ζ protein. Three molecules, derived from the endogenous intermediate isatin termed, FZIB-35, FZIB-36 and FZIB-38 were identified as novel inhibitors which shows significant interaction with 14-3-3ζ. A mutation in the binding groove of 14-3-3ζ, (K49E), almost abolishes the binding of these compounds to 14-3-3ζ protein. To exclude the probability of false positives, two more purified proteins (PtpA and BirA) were also tested. Furthermore, in order to confirm the binding pocket specificity, competition assay against R18 peptide was also carried out on presented platform. We show that SMMs in combination with SPRi are a powerful method to identify lead compounds in high throughput manner without the need to develop an activity based assay.

Probing small-molecule microarrays with tagged proteins in cell lysates.

The technique of small-molecule microarray (SMM) screening is based on the ability of small molecules to bind to various soluble proteins. This type of interaction is easily detected by the presence of a fluorescence signal produced by labeled antibodies that specifically recognize a unique sequence (tag) present on the target protein. The fluorescent signal intensity values are determined based on signal-to-noise ratios (SNRs). SMM screening is a high-throughput, unbiased method that can rapidly identify novel direct ligands for various protein targets. This binding-based assay format is generally applicable to most proteins, but it is especially useful for protein targets that do not possess an enzymatic activity. SMMs enable screening a protein in a purified form or in the context of a cellular lysate, likely providing a more physiologically relevant screening environment.

Abstract 1629: Identification and biological characterization of a novel class of small molecules to inhibit c-myc transcription

Abstract The transcription factor c-myc, deregulated in ∼70% of all cancer cases, facilitates tumor initiation and promotion most frequently in colon, breast, lung, ovarian, and lymphoid malignancies, and acts as a universal amplifier of aberrant growth-related cell signaling. Due to its helix-loop helix topology, short protein half-life, and rapid replenishment within the cancer cell, c-myc was long considered to be ‘undruggable.’ Recently, much attention has focused on inhibiting c-myc either through indirect pathway modulation, or on the transcriptional level. 85-90% of c-myc gene expression is controlled by a G-quadruplex (G4) DNA structure found in the nuclease hypersensitive element III(1) region (NHEIII1) of its promoter, which, when stabilized with a chemical agent, would theoretically inhibit myc transcription by preventing normal polymerase function. In this study, we report the identification and biological characterization of a novel chemical class to directly bind the c-myc G4 DNA. The chemical motif discussed here was identified from a highly selective (hit rate of 0.16%, with few false positives) small molecule microarray screen of 20,000 compounds. A panel of the 12 most promising hits was chosen for biological and biochemical secondary validation in a variety of in vitro and cell based assays to confirm mechanism of action and functional effects in biological systems. Of note, numerous compounds from the initial panel of hits exhibited effects with a range of potency in comparison with untreated and non quadruplex forming nucleic acid controls, thereby validating the original screening method. For prioritization, hits were further evaluated for their capacity to preferentially induce myc-dependent cell death in a tetracycline controlled c-myc stable cell line. A top lead was identified, and structure-activity relationship was preliminarily evaluated with a small panel of analogues, leading to a final compound with three times greater potency. The chosen compound exhibited strong dose-dependent cell killing activity, in human multiple myeloma with high levels of c-myc, with an IC50 in the single digit micromolar range. Additionally, the compound reduced c-myc gene expression in a series of B cell neoplasms to near the limit of detection, both transcriptionally and translationally. The mechanism of action was confirmed in a Burkitt's Lymphoma model through a quadruplex promoter-specific comparative analysis of myc transcript copy numbers in the presence or absence of treatment. Continuing studies will focus on further structural optimization of the ligand, a thorough examination of its genome-wide effects, and tolerability in animal models. Citation Format: Kenneth M. Felsenstein, John K. Simmons, Peter Gareiss, Beverly A. Mock, John ‘Jay’ S. Schneekloth. Identification and biological characterization of a novel class of small molecules to inhibit c-myc transcription. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1629. doi:10.1158/1538-7445.AM2014-1629

Screening and discovery of nitro-benzoxadiazole compounds activating epidermal growth factor receptor (EGFR) in cancer cells

Peptide ligand-induced dimerization of the extracellular region of the epidermal growth factor receptor (sEGFR) is central to the signal transduction of many cellular processes. A small molecule microarray screen has been developed to search for non-peptide compounds able to bind to sEGFR. We describe the discovery of nitro-benzoxadiazole (NBD) compounds that enhance tyrosine phosphorylation of EGFR and thereby trigger downstream signaling pathways and other receptor tyrosine kinases in cancer cells. The protein phosphorylation profile in cells exposed to NBD compounds is to some extent reminiscent of the profile induced by the cognate ligand. Experimental studies indicate that the small compounds bind to the dimerization domain of sEGFR, and generate stable dimers providing allosteric activation of the receptor. Moreover, receptor phosphorylation is associated with inhibition of PTP-1B phosphatase. Our data offer a promising paradigm for investigating new aspects of signal transduction mediated by EGFR in cancer cells exposed to electrophilic NBD compounds.

A HaloTag-Based Small Molecule Microarray Screening Methodology with Increased Sensitivity and Multiplex Capabilities

Small Molecule Microarrays (SMMs) represent a general platform for screening small molecule-protein interactions independent of functional inhibition of target proteins. In an effort to increase the scope and utility of SMMs, we have modified the SMM screening methodology to increase assay sensitivity and facilitate multiplex screening. Fusing target proteins to the HaloTag protein allows us to covalently prelabel fusion proteins with fluorophores, leading to increased assay sensitivity and an ability to conduct multiplex screens. We use the interaction between FKBP12 and two ligands, rapamycin and ARIAD's "bump" ligand, to show that the HaloTag-based SMM screening methodology significantly increases assay sensitivity. Additionally, using wild type FKBP12 and the FKBP12 F36V mutant, we show that prelabeling various protein isoforms with different fluorophores allows us to conduct multiplex screens and identify ligands to a specific isoform. Finally, we show this multiplex screening technique is capable of identifying ligands selective for a specific PTP1B isoform using a 20,000 compound screening deck.