scholarly journals Partitioning of cancer therapeutics in nuclear condensates.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 3131-3131
Author(s):  
Isaac Klein ◽  
Ann Boija ◽  
Lena Afeyan ◽  
Susana Hawken ◽  
Mengyang Fan ◽  
...  
Keyword(s):  
Physicochemical Properties ◽  
Small Molecule ◽  
Cancer Therapeutics ◽  
Therapeutic Index ◽  
Regulatory Elements ◽  
Specific Protein ◽  
Target Engagement ◽  
Membrane Bound ◽  
In Cells

3131 Background: The molecules of the cell are compartmentalized into membrane- and non-membrane-bound organelles. Many non-membrane-bound organelles are phase-separated biomolecular condensates with distinct physicochemical properties that can absorb and concentrate specific proteins and nucleic acids involved in discrete biochemical processes. We reasoned that selective condensate partitioning might also occur with small molecule drugs whose targets occur within condensates, and that the therapeutic index and efficacy of such compounds might therefore relate to their ability to partition into condensates. Methods: To study the behavior of drugs within condensates, these were modeled in cells and in vitro with purified proteins and visualized by fluorescent confocal microscopy. The functional outcomes of condensate partitioning were queried in cells. Results: We found that cisplatin, tamoxifen, JQ1, THZ1, and mitoxantrone are concentrated in specific protein condensates in vitro, and that this occurs through physicochemical properties independent of the drug target. A screen of a chemically diverse fluorescent probes and mutant-protein condensates demonstrated that pi-system interactions between aromatic moieties in the protein and small molecule govern concentration in condensates. These results show that clinically important drugs partition into specific protein condensates in vitro by virtue of defined chemical properties, thereby altering their local concentration. In vitro droplet assays revealed that cisplatin is selectively concentrated in transcriptional condensates, and that this ability is required for efficient platination of target DNA. In cell studies revealed that cisplatin preferentially targets DNA contained within MED1 condensates, and disrupts the genetic regulatory elements that compose phase-separated transcriptional condensates. Live cell imaging demonstrated that transcriptional condensates are dissolved by cisplatin, whereas other condensates remain intact. Conclusions: Our results show that antineoplastic drugs partition selectively into biomolecular condensates, that this can occur through physicochemical properties independent of their molecular targets, and that drug activity may occur through condensate-related mechanisms. These results have implications for development of efficacious cancer therapeutics; effective target engagement will depend on factors such as drug partitioning in condensates. Assays of the type described here may thus help optimize condensate partitioning, target engagement, and the therapeutic index of drugs for cancer treatment.

scholarly journals Partitioning of cancer therapeutics in nuclear condensates

Science ◽  
2020 ◽  
Vol 368 (6497) ◽  
pp. 1386-1392 ◽  
Author(s):  
Isaac A. Klein ◽  
Ann Boija ◽  
Lena K. Afeyan ◽  
Susana Wilson Hawken ◽  
Mengyang Fan ◽  
...  
Keyword(s):  
Physicochemical Properties ◽  
Small Molecules ◽  
Tumor Cells ◽  
Drug Target ◽  
Cancer Therapeutics ◽  
Specific Protein ◽  
Antineoplastic Drugs ◽  
Disease Therapy ◽  
Drug Partitioning

The nucleus contains diverse phase-separated condensates that compartmentalize and concentrate biomolecules with distinct physicochemical properties. Here, we investigated whether condensates concentrate small-molecule cancer therapeutics such that their pharmacodynamic properties are altered. We found that antineoplastic drugs become concentrated in specific protein condensates in vitro and that this occurs through physicochemical properties independent of the drug target. This behavior was also observed in tumor cells, where drug partitioning influenced drug activity. Altering the properties of the condensate was found to affect the concentration and activity of drugs. These results suggest that selective partitioning and concentration of small molecules within condensates contributes to drug pharmacodynamics and that further understanding of this phenomenon may facilitate advances in disease therapy.

scholarly journals Cooperative Assembly of an hnRNP Complex Induced by a Tissue-Specific Homolog of Polypyrimidine Tract Binding Protein

2000 ◽  
Vol 20 (20) ◽  
pp. 7463-7479 ◽  
Author(s):  
Vadim Markovtsov ◽  
Julia M. Nikolic ◽  
Joseph A. Goldman ◽  
Christoph W. Turck ◽  
Min-Yuan Chou ◽  
...  
Keyword(s):  
Binding Protein ◽  
Regulatory Protein ◽  
Regulatory Elements ◽  
Specific Protein ◽  
Regulatory Sequence ◽  
Polypyrimidine Tract ◽  
Tissue Specific ◽  
Polypyrimidine Tract Binding ◽  
Polypyrimidine Tract Binding Protein

ABSTRACT Splicing of the c-src N1 exon in neuronal cells depends in part on an intronic cluster of RNA regulatory elements called the downstream control sequence (DCS). Using site-specific cross-linking, RNA gel shift, and DCS RNA affinity chromatography assays, we characterized the binding of several proteins to specific sites along the DCS RNA. Heterogeneous nuclear ribonucleoprotein (hnRNP) H, polypyrimidine tract binding protein (PTB), and KH-type splicing-regulatory protein (KSRP) each bind to distinct elements within this sequence. We also identified a new 60-kDa tissue-specific protein that binds to the CUCUCU splicing repressor element of the DCS RNA. This protein was purified, partially sequenced, and cloned. The new protein (neurally enriched homolog of PTB [nPTB]) is highly homologous to PTB. Unlike PTB, nPTB is enriched in the brain and in some neural cell lines. Although similar in sequence, nPTB and PTB show significant differences in their properties. nPTB binds more stably to the DCS RNA than PTB does but is a weaker repressor of splicing in vitro. nPTB also greatly enhances the binding of two other proteins, hnRNP H and KSRP, to the DCS RNA. These experiments identify specific cooperative interactions between the proteins that assemble onto an intricate splicing-regulatory sequence and show how this hnRNP assembly is altered in different cell types by incorporating different but highly related proteins.

scholarly journals New tools for carbohydrate sulphation analysis: Heparan Sulphate 2-O-sulphotranserase (HS2ST) is a target for small molecule protein kinase inhibitors

2018 ◽  
Author(s):  
Dominic P Byrne ◽  
Yong Li ◽  
Krithika Ramakrishnan ◽  
Igor L Barsukov ◽  
Edwin A Yates ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Small Molecule ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Heparan Sulphate ◽  
Protein Kinase Inhibitors ◽  
Fluorescent Substrate ◽  
Shift Analysis ◽  
In Cells

ABSTRACTSulphation of carbohydrate residues occurs on a variety of glycans destined for secretion, and this modification is essential for efficient matrix-based signal transduction. Heparan sulphate (HS) glycosaminoglycans control physiological functions ranging from blood coagulation to cell proliferation. HS biosynthesis involves membrane-bound Golgi sulphotransferases, including heparan sulphate 2-O-sulphotransferase (HS2ST), which transfers sulphate from the co-factor PAPS (3’-phosphoadenosine 5’-phosphosulphate) to the 2-Oposition of α-L-iduronate in the maturing oligosaccharide chain. The current lack of simple non-radioactive enzyme assays that can be used to quantify the levels of carbohydrate sulphation hampers kinetic analysis of this process and the discovery of HS2ST inhibitors. In this paper, we describe a new procedure for thermal shift analysis of purified HS2ST. Using this approach, we quantify HS2ST-catalyzed oligosaccharide sulphation using a novel synthetic fluorescent substrate and screen the Published Kinase Inhibitor Set (PKIS), to evaluate compounds that inhibit catalysis. We report the susceptibility of HS2ST to a variety of cell permeable compoundsin vitro, including polyanionic polar molecules, the protein kinase inhibitor rottlerin and oxindole-based RAF kinase inhibitors. In a related study, published back-to-back with this article, we demonstrate that Tyrosyl Protein Sulpho Tranferases (TPSTs) are also inhibited by a variety of protein kinase inhibitors. We propose that appropriately validated small molecule compounds could become new tools for rapid inhibition of glycan (and protein) sulphation in cells, and that protein kinase inhibitors might be repurposed or redesigned for the specific inhibition of HS2ST.SUMMARY STATEMENTWe report that HS2ST, which is a PAPS-dependent glycan sulphotransferase, can be assayed using a variety of novel biochemical procedures, including a non-radioactive enzyme-based assay that detects glycan substrate sulphation in real time. HS2ST activity can be inhibited by different classes of compounds, including known protein kinase inhibitors, suggesting new approaches to evaluate the roles of HS2ST-dependent sulphation with small molecules in cells.

scholarly journals Induction of Fetal Hemoglobin By FTX6058, a Novel Small Molecule Development Candidate

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 1-1
Author(s):  
Peter Rahl ◽  
Ivan Efremov ◽  
Billy Stuart ◽  
Keqiang Xie ◽  
Mark Roth ◽  
...  
Keyword(s):  
Small Molecule ◽  
Plasma Concentrations ◽  
Fetal Hemoglobin ◽  
Target Engagement ◽  
Publicly Traded ◽  
In Vivo Models ◽  
Α Subunit ◽  
The Past

Red blood cell disorders like Sickle Cell Disease (SCD) and β-thalassemias are caused by mutations within the gene for the hemoglobin β (HBβ) subunit. A fetal ortholog of HBβ, hemoglobin γ (HBγ) can prevent or reduce disease-related pathophysiology in these disorders by forming nonpathogenic complexes with the required hemoglobin α-subunit. Globin expression is developmentally regulated, with a reduction in production of the fetal ortholog (γ)occurring shortly after birth and a concomitant increase in the levels of the adult ortholog (β). It has been postulated that maintaining expression of the anti-sickling γ ortholog may be of therapeutic benefit in children and adults with SCD. Indeed, individuals with the SCD mutation who also have genetic variants that maintain HBγ expression at clinically meaningful levels do not present with SCD-related symptoms. Parallel target identification efforts using CRISPR and the Fulcrum proprietary, annotated chemical probe screening set in HUDEP2 cells identified a protein complex as a key regulator of HbF expression. Structure-guided medicinal chemistry optimization led to the design of FTX-6058, a novel, potent and selective small molecule with desirable DMPK properties suitable for clinical testing. FTX-6058 treatment of differentiated primary CD34+ cells from multiple healthy donors demonstrated target engagement and potent upregulation of HBG1/2 mRNA and HbF protein. Across multiple healthy and SCD donors, FTX-6058 treatment resulted in a clinically desirable globin profile (e.g., up to 30% absolute HbF) accompanied by pancellular HbF expression, resembling the phenotype of SCD mutation carriers with hereditary persistence of fetal hemoglobin. FTX-6058 demonstrated a superior pharmacological profile relative to hydroxyurea and other small molecule compounds whose putative mechanism of action is to induce HbF. FTX-6058 treatment resulted in robust target engagement and subsequent elevation of the endogenous mouse Hbb-bh1 mRNA in wildtype CD-1 mice and, importantly, also elevation of the human HBG1 mRNA and HbF protein in the Townes SCD mouse model. Preclinical studies using a variety of in vitro and in vivo models have demonstrated the potential of FTX-6058 as a novel HbF-inducing small molecule that could be beneficial to patients with SCD and β-thalassemias. FTX-6058 was shown to be potent and selective in vitro, was well tolerated and elicited a desirable exposure-response relationship in multiple preclinical rodent models with once-a-day oral dosing and at plasma concentrations predicted to be achievable in patients. IND enabling studies for FTX-6058 have been completed. Disclosures Rahl: Fulcrum Therapeutics: Ended employment in the past 24 months. Efremov:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Stuart:Fulcrum Therapeutics: Current Employment, Current equity holder in publicly-traded company. Xie:Fulcrum Therapeutics: Current Employment. Roth:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Barnes:Fulcrum Therapeutics: Ended employment in the past 24 months. Appiah:Fulcrum Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Peters:Fulcrum Therapeutics: Current Employment. Li:Fulcrum Therapeutics: Ended employment in the past 24 months. Kazmirski:Fulcrum Therapeutics: Ended employment in the past 24 months. Bruno:Fulcrum Therapeutics: Current Employment. Stickland:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Ronco:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Cadavid:Fulcrum Therapeutics: Current Employment, Current equity holder in publicly-traded company. Thompson:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Wallace:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Moxham:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company.

Golgi recruitment assay for visualizing small-molecule ligand–target engagement in cells

2020 ◽  
Vol 56 (57) ◽  
pp. 7961-7964
Author(s):  
Sachio Suzuki ◽  
Masahiro Ikuta ◽  
Tatsuyuki Yoshii ◽  
Akinobu Nakamura ◽  
Keiko Kuwata ◽  
...  
Keyword(s):  
Small Molecule ◽  
Living Cells ◽  
New Method ◽  
Target Engagement ◽  
Small Molecule Ligand ◽  
In Cells

A Golgi recruitment (G-REC) assay is developed as a new method for visualizing small-molecule ligand–target engagement in living cells.

scholarly journals Small-Molecule Target Engagement in Cells

2016 ◽  
Vol 23 (4) ◽  
pp. 435-441 ◽  
Author(s):  
Marc Schürmann ◽  
Petra Janning ◽  
Slava Ziegler ◽  
Herbert Waldmann
Keyword(s):  
Small Molecule ◽  
Target Engagement ◽  
In Cells

scholarly journals Translation of the intrinsically disordered protein α-synuclein is inhibited by a small molecule targeting its structured mRNA

2020 ◽  
Vol 117 (3) ◽  
pp. 1457-1467 ◽  
Author(s):  
Peiyuan Zhang ◽  
Hye-Jin Park ◽  
Jie Zhang ◽  
Eunsung Junn ◽  
Ryan J. Andrews ◽  
...  
Keyword(s):  
Small Molecules ◽  
Small Molecule ◽  
Intrinsically Disordered Proteins ◽  
Lewy Bodies ◽  
Disordered Proteins ◽  
Lewy Neurites ◽  
Protein Levels ◽  
Intrinsically Disordered ◽  
In Cells

Many proteins are refractory to targeting because they lack small-molecule binding pockets. An alternative to drugging these proteins directly is to target the messenger (m)RNA that encodes them, thereby reducing protein levels. We describe such an approach for the difficult-to-target protein α-synuclein encoded by the SNCA gene. Multiplication of the SNCA gene locus causes dominantly inherited Parkinson’s disease (PD), and α-synuclein protein aggregates in Lewy bodies and Lewy neurites in sporadic PD. Thus, reducing the expression of α-synuclein protein is expected to have therapeutic value. Fortuitously, the SNCA mRNA has a structured iron-responsive element (IRE) in its 5′ untranslated region (5′ UTR) that controls its translation. Using sequence-based design, we discovered small molecules that target the IRE structure and inhibit SNCA translation in cells, the most potent of which is named Synucleozid. Both in vitro and cellular profiling studies showed Synucleozid directly targets the α-synuclein mRNA 5′ UTR at the designed site. Mechanistic studies revealed that Synucleozid reduces α-synuclein protein levels by decreasing the amount of SNCA mRNA loaded into polysomes, mechanistically providing a cytoprotective effect in cells. Proteome- and transcriptome-wide studies showed that the compound’s selectivity makes Synucleozid suitable for further development. Importantly, transcriptome-wide analysis of mRNAs that encode intrinsically disordered proteins revealed that each has structured regions that could be targeted with small molecules. These findings demonstrate the potential for targeting undruggable proteins at the level of their coding mRNAs. This approach, as applied to SNCA, is a promising disease-modifying therapeutic strategy for PD and other α-synucleinopathies.

scholarly journals The small molecule H89 inhibits Chlamydia inclusion growth and production of infectious progeny

2021 ◽  
Author(s):  
Karissa J. Muñoz ◽  
Kevin Wang ◽  
Lauren M. Sheehan ◽  
Ming Tan ◽  
Christine Sütterlin
Keyword(s):  
Protein Kinases ◽  
Small Molecule ◽  
Specific Protein ◽  
Human Infection ◽  
Developmental Cycle ◽  
Growth State ◽  
Reticulate Body ◽  
Fold Reduction ◽  
Membrane Bound ◽  
Infectious Form

Chlamydia is an obligate intracellular bacterium and the most common reportable cause of human infection in the U.S. This pathogen proliferates inside a eukaryotic host cell, where it resides within a membrane-bound compartment called the chlamydial inclusion. It has an unusual developmental cycle, marked by conversion between a replicating form, the reticulate body (RB), and an infectious form, the elementary body (EB). We found that the small molecule H89 slowed inclusion growth and decreased overall RB replication by 2-fold, but caused a 25-fold reduction in infectious EBs. This disproportionate effect on EB production was mainly due to a defect in RB-to-EB conversion and not to the induction of chlamydial persistence, which is an altered growth state. Although H89 is a known inhibitor of specific protein kinases and vesicular transport to and from the Golgi, it did not cause these anti-chlamydial effects by blocking the protein kinases PKA or PKC, or by inhibiting protein or lipid transport. H89 is thus a novel anti-chlamydial compound that has a unique combination of effects on the intracellular Chlamydia infection.

Sign in / Sign up

Export Citation Format

Share Document