Discovery and Optimization of Pyrrolopyrimidine Derivatives as Selective Disruptors of the Perinucleolar Compartment, a Marker of Tumor Progression toward Metastasis

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Journal of Medicinal Chemistry, Copyright © 2022 American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/acs.jmedchem.2c00204.The perinucleolar compartment (PNC) is a dynamic subnuclear body found at the periphery of the nucleolus. The PNC is enriched with RNA transcripts and RNA-binding proteins, reflecting different states of genome organization. PNC prevalence positively correlates with cancer progression and metastatic capacity, making it a useful marker for metastatic cancer progression. A high-throughput, high-content assay was developed to identify novel small molecules that selectively reduce PNC prevalence in cancer cells. We identified and further optimized a pyrrolopyrimidine series able to reduce PNC prevalence in PC3M cancer cells at submicromolar concentrations without affecting cell viability. Structure–activity relationship exploration of the structural elements necessary for activity resulted in the discovery of several potent compounds. Analysis of in vitro drug-like properties led to the discovery of the bioavailable analogue, metarrestin, which has shown potent antimetastatic activity with improved survival in rodent models and is currently being evaluated in a first-in-human phase 1 clinical trial

A High Throughput Screening Assay System for the Identification of Small Molecule Inhibitors of <i>gsp</i>

<div><p>Mis-sense mutations in the α-subunit of the G-protein, G_sα, cause fibrous dysplasia of bone/McCune-Albright syndrome. The biochemical outcome of these mutations is constitutively active G_sα and increased levels of cAMP. The aim of this study was to develop an assay system that would allow the identification of small molecule inhibitors specific for the mutant G_sα protein, the so-called <i>gsp</i> oncogene. Commercially available Chinese hamster ovary cells were stably transfected with either wild-type (WT) or mutant G_sα proteins (R201C and R201H). Stable cell lines with equivalent transfected G_sα protein expression that had relatively lower (WT) or higher (R201C and R201H) cAMP levels were generated. These cell lines were used to develop a fluorescence resonance energy transfer (FRET)–based cAMP assay in 1536-well microplate format for high throughput screening of small molecule libraries. A small molecule library of 343,768 compounds was screened to identify modulators of <i>gsp</i> activity. A total of 1,356 compounds with inhibitory activity were initially identified and reconfirmed when tested in concentration dose responses. Six hundred eighty-six molecules were selected for further analysis after removing cytotoxic compounds and those that were active in forskolin-induced WT cells. These molecules were grouped by potency, efficacy, and structural similarities to yield 22 clusters with more than 5 of structurally similar members and 144 singleton molecules. Seven chemotypes of the major clusters were identified for further testing and analyses.</p></div

Compound Identification Flow Chart.

<p>Depicted is a flow chart of the assays, filtering, and analyses that were performed to ultimately identify the 7 chemotypes of clusters of molecules that have been selected for further study.</p

Summary of Confirmation Hits.

<p>Summary of Confirmation Hits.</p

Clustering Analysis.

<p>Active compounds were clustered based on structural similarity to identify common chemotypes using LeadScope (Leadscope Hosted Client, Leadscope Inc., Columbus, OH). The results show a diversity of structural clusters, with 22 distinct clusters with more than 5 members. (A) Representative compounds from each of the most prominent 7 clusters are shown. (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090766#pone.0090766.s015" target="_blank">Table S5</a>, Cluster Analysis Compounds with Link for a complete list of the 102 molecules in the 7 clusters, their structures, IC₅₀, and active link to the complete PubChem description). Their common structural scaffolds are highlighted in red. These scaffolds are highly polar, including thiazole, triazole, and hydrozide-based derivatives. Another common structural feature is that these small molecules share a linear molecular shape, which suggests that they might compete with GTP at the active site of the G protein. (B) Inhibition-concentration curves for 7 selected compounds, one from each cluster, together with the IC₅₀ for each compound are shown.</p